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Dr. Weissman directs a research group consisting of graduate students, medical student-scientists, and postdoctoral fellows, all of whom study stem cell biology and regenerative medicine. He has trained and supervised hundreds of students and fellows, authored more than 750 scientific articles and has numerous awards and honorary degrees for his research accomplishments. He is an elected member of the National Academy of Sciences, the Institute of Medicine, the American Academy of Arts and Sciences, the Amerian Philosophical Society, and many other societies. He is past president of the American Association of Immunologists [1994] and the International Society of Stem Cell Research [2009]. Dr. Weissman is an expert in the field of hematopoiesis, leukemia, and hematopoietic stem cells [HSC], and most recently, the clonal events leading from HSC to leukemia stem cells.. His research also encompasses the phylogeny and developmental biology of the cells that make up the blood-forming and immune systems. He has a laboratory at Hopkins Marine Station of Stanford University, where he studies the histocompatibility systems in colonial protochordate, a system which he proposed evolved to prevent predatory germline stem cell lineages from passing from one individual to another in multi-individual colonies that share a common extracorporeal blood vascular system; only histocompatible stem cells can colonize allogeneic natural parabionts. His laboratory was first to identify and isolate the blood-forming stem cell [HSC] from mice, and has defined, by lineage analysis, the stages of development between the stem cells and mature progeny. His laboratories have also discovered the human HSC, a human brain-forming stem cell population, mouse skeletal muscle stem cells, and an osteochondral stem cell in mice. He has worked in the area of cancer since 1977, and is a leader in the field of cancer stem cell biology. In recent years his work has included studying the potential of CD47 as a cancer therapeutic and identifying cancer stem cells from a variety of blood and solid cancers. He and his colleagues have found that CD47, a ‘don’t eat me signal’ is highly expressed beginning in the latter stages of progression of cancer stem cells from the benign to the highly malignant state, and this counteracts ‘eat me’ signals on preneoplastic and highly malignant cancer cells, presumably as part of the evolution of cancer clones driven by self-renewing subsets of cells in the cancer. This research brings into focus the primary role of phagocytic cells such as macrophages of the innate immune system, in tumor surveillance. Dr. Weissman was a founder of three companies, SyStemix, Cellerant, and Stem Cells, Inc., all focused on bringing stem cell therapies into the clinic, and earlier was on the founding SABs of Amgen, DNAX, and T Cell Sciences.

Clinical Trials

We hypothesize that all human malignancies harbour a subpopulation of tumor initiating
cells/cancer stem cells (CSCs) that drives tumor development and potentially recurrence or
metastasis of the disease. The primary aim of this study is to develop strategies for
prospective isolation/enrichment of CSCs from human tumors of different tissue origins. In
addition, we will characterize the signaling pathways and/or tumor specific antigens that are
specific for CSCs, in order to specifically target these CSCs as the endpoint of this study.

Stanford is currently not accepting patients for this trial.For more information, please contact Linda Quinn, 650-723-6520.

Microarray Analysis of Gene Expression and Identification of Progenitor Cells in Lung CarcinomaRecruiting

This study will investigate gene expression profiles in normal human lung tissue, lung
carcinoma and metastatic tumor to the lung. The expression of up to 20,000 genes in a given
lung tissue sample will be examined by cDNA microarray analysis and compared to normal lung
tissue. In addition, we hope to identify a particular subset of lung cancer cells with an
enhanced capacity for proliferation and self-renewal , analogous to the stem cells recently
identified for certain types of leukemia, breast cancer and brain tumors.

Abstract

Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. Here, we sought to optimize noninvasive immuno-PET imaging of human programmed death-ligand 1 (PD-L1) expression, in a preclinical model, using a small high-affinity engineered protein scaffold (HAC-PD1). Six HAC-PD1 radiotracer variants were developed and used in preclinical imaging and biodistribution studies to assess their ability to detect human PD-L1 expression in vivo. Radiotracer design modifications included chelate, glycosylation, and radiometal. HACA-PD1 was adopted as the naming convention for aglycosylated tracer variants. NOD scid ?-(NSG) mice were inoculated with subcutaneous tumors engineered to either be constitutively positive (CT26 hPD-L1) or be negative (?mPD-L1 CT26) for human PD-L1 expression. When the tumors had grown to an average size of 1 cm in diameter, mice were injected with 0.75-2.25 MBq (?10 ?g) of an engineered radiotracer variant and imaged. At 1 h after injection, organs were harvested for biodistribution. Of the practical immuno-PET tracer modifications considered, glycosylation was the most prominent design factor affecting tracer uptake, specificity, and clearance. In imaging studies, aglycosylated (64)Cu-NOTA-HACA-PD1 most accurately visualized human PD-L1 expression in vivo. We reasoned that because of the scaffold's small size (14 kDa), its pharmacokinetics may be suitable for labeling with the short-lived and widely clinically available radiometal (68)Ga. At 1 h after injection, (68)Ga-NOTA-HACA-PD1 and (68)Ga-DOTA-HACA-PD1 exhibited promising target-to-background ratios in ex vivo biodistribution studies (12.3 and 15.2 tumor-to-muscle ratios, respectively). Notably, all HAC-PD1 radiotracer variants enabled much earlier detection of human PD-L1 expression (1 h after injection) than previously reported radiolabeled antibodies (>24 h after injection). This work provides a template for assessing immuno-PET tracer design parameters and supports the translation of small engineered protein radiotracers for imaging human immune checkpoints.

Abstract

The contribution of the different waves and sites of developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre. Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%-40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1(-/-) concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5-13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization.

Abstract

Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

Abstract

The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRP? axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.

Abstract

The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271(+) melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2(+)/VEGFR1(+)), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271(+) melanoma cells represents a powerful therapeutic approach against metastatic melanoma.

Abstract

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-? as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

Abstract

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRP?), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRP? using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRP? axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

Abstract

In a primitive chordate model of natural chimerism, one chimeric partner is often eliminated in a process of allogeneic resorption. Here, we identify the cellular framework underlying loss of tolerance to one partner within a natural Botryllus schlosseri chimera. We show that the principal cell type mediating chimeric partner elimination is a cytotoxic morula cell (MC). Proinflammatory, developmental cell death programs render MCs cytotoxic and, in collaboration with activated phagocytes, eliminate chimeric partners during the "takeover" phase of blastogenic development. Among these genes, the proinflammatory cytokine IL-17 enhances cytotoxicity in allorecognition assays. Cellular transfer of FACS-purified MCs from allogeneic donors into recipients shows that the resorption response can be adoptively acquired. Transfer of 1 × 10(5) allogeneic MCs eliminated 33 of 78 (42%) recipient primary buds and 20 of 76 (20.5%) adult parental adult organisms (zooids) by 14 d whereas transfer of allogeneic cell populations lacking MCs had only minimal effects on recipient colonies. Furthermore, reactivity of transferred cells coincided with the onset of developmental-regulated cell death programs and disproportionately affected developing tissues within a chimera. Among chimeric partner "losers," severe developmental defects were observed in asexually propagating tissues, reflecting a pathologic switch in gene expression in developmental programs. These studies provide evidence that elimination of one partner in a chimera is an immune cell-based rejection that operates within histocompatible pairs and that maximal allogeneic responses involve the coordination of both phagocytic programs and the "arming" of cytotoxic cells.

Abstract

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Abstract

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

Abstract

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Abstract

Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

Abstract

This position statement originated from a working group meeting convened on April 15, 2015, by the NHLBI and incorporates follow-up contributions by the participants as well as other thought leaders subsequently consulted, who together represent research fields relevant to all branches of the NIH. The group was deliberately composed not only of individuals with a current research emphasis in the glycosciences, but also of many experts from other fields, who evinced a strong interest in being involved in the discussions. The original goal was to discuss the value of creating centers of excellence for training the next generation of biomedical investigators in the glycosciences. A broader theme that emerged was the urgent need to bring the glycosciences back into the mainstream of biology by integrating relevant education into the curricula of medical, graduate, and postgraduate training programs, thus generating a critical sustainable workforce that can advance the much-needed translation of glycosciences into a more complete understanding of biology and the enhanced practice of medicine.

Abstract

The presence, characteristics, and potential clinical relevance of neural progenitor populations within the neural placodes of myelomeningocele patients remain to be studied. Neural stem cells are known to reside adjacent to ependyma-lined surfaces along the central nervous system axis.Given such neuroanatomic correlation and regenerative capacity in fetal development, we assessed myelomeningocele-derived neural placode tissue as a potentially novel source of neural stem and progenitor cells.Nonfunctional neural placode tissue was harvested from infants during the surgical repair of myelomeningocele and subsequently further analyzed by in vitro studies, flow cytometry, and immunofluorescence. To assess lineage potential, neural placode-derived neurospheres were subjected to differential media conditions. Through assessment of platelet-derived growth factor receptor ? (PDGFR?) and CD15 cell marker expression, Sox2+Olig2+ putative oligodendrocyte progenitor cells were successfully isolated.PDGFR?CD15 cell populations demonstrated the highest rate of self-renewal capacity and multipotency of cell progeny. Immunofluorescence of neural placode-derived neurospheres demonstrated preferential expression of the oligodendrocyte progenitor marker, CNPase, whereas differentiation to neurons and astrocytes was also noted, albeit to a limited degree.Neural placode tissue contains multipotent progenitors that are preferentially biased toward oligodendrocyte progenitor cell differentiation and presents a novel source of such cells for use in the treatment of a variety of pediatric and adult neurological disease, including spinal cord injury, multiple sclerosis, and metabolic leukoencephalopathies.

Abstract

The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.

Abstract

Determining the developmental pathway leading to erythrocytes and being able to isolate their progenitors are crucial to understanding and treating disorders of red cell imbalance such as anemia, myelodysplastic syndrome, and polycythemia vera. Here we show that the human erythrocyte progenitor (hEP) can be prospectively isolated from adult bone marrow. We found three subfractions that possessed different expression patterns of CD105 and CD71 within the previously defined human megakaryocyte/erythrocyte progenitor (hMEP; Lineage(-) CD34(+) CD38(+) IL-3R?(-) CD45RA(-)) population. Both CD71(-) CD105(-) and CD71(+) CD105(-) MEPs, at least in vitro, still retained bipotency for the megakaryocyte (MegK) and erythrocyte (E) lineages, although the latter subpopulation is skewed in differentiation toward the erythroid lineage. Notably, the proliferative and differentiation output of the CD71(intermediate(int)/+) CD105(+) subset of cells within the MEP population was completely restricted to the erythroid lineage with the loss of MegK potential. CD71(+) CD105(-) MEPs are erythrocyte-biased MEPs (E-MEPs) and CD71(int/+) CD105(+) cells are EPs. These previously unclassified populations may facilitate further understanding of the molecular mechanisms governing human erythroid development and serve as potential therapeutic targets in disorders of the erythroid lineage.

Abstract

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-V?8.2 in absence of anti-C? antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface V?8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of V?8.2(+) C?(-) are found in several lymphoid sites, and among the lineage-negative (Lin(-) ) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin(-) V?8.2(+) C?(-) BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin(-) V?8.2(+) C?(-) BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

Stem cells are units of natural selection for tissue formation, for germline development, and in cancer developmentPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWeissman, I. L.2015; 112 (29): 8922-8928

Abstract

It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer.

Abstract

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein ? (SIRP?). Molecules that antagonize the CD47-SIRP? interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRP? variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRP? pathway by engineering high affinity CD47 variants that target SIRP?, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRP?. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRP? alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRP? on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRP? compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

Abstract

Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

Abstract

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.

Abstract

Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

Abstract

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fc? receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein ? (SIRP?) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

Abstract

Current methods for the isolation of fibroblasts require extended ex vivo manipulation in cell culture. As a consequence, prior studies investigating fibroblast biology may fail to adequately represent cellular phenotypes in vivo. To overcome this problem, we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of adult mice that bypasses the need for cell culture, thereby preserving the physiological, transcriptional, and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional programs and the surface expression of 176 CD markers in cultured versus uncultured fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest for investigations of fibroblast biology.

Abstract

Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein ? to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

Abstract

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Abstract

The generation of pluripotent stem cells by somatic cell nuclear transfer (SCNT) has recently been achieved in human cells and sparked new interest in this technology. The authors reporting this methodical breakthrough speculated that SCNT would allow the creation of patient-matched embryonic stem cells, even in patients with hereditary mitochondrial diseases. However, herein we show that mismatched mitochondria in nuclear-transfer-derived embryonic stem cells (NT-ESCs) possess alloantigenicity and are subject to immune rejection. In a murine transplantation setup, we demonstrate that allogeneic mitochondria in NT-ESCs, which are nucleus-identical to the recipient, may trigger an adaptive alloimmune response that impairs the survival of NT-ESC grafts. The immune response is adaptive, directed against mitochondrial content, and amenable for tolerance induction. Mitochondrial alloantigenicity should therefore be considered when developing therapeutic SCNT-based strategies.

Abstract

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a bone marrow cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13 and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs) as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

Abstract

Epicardial cells on the heart's surface give rise to coronary artery smooth muscle cells (caSMCs) located deep in the myocardium. However, the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries. Here, we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes, mural cells associated with microvessels, and that these cells are present in adults. During development following the onset of blood flow, pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1. Deletion of Notch3 disrupts caSMC differentiation. Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature, but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling. Our data are the first demonstration that pericytes are progenitors for smooth muscle, and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease.

Abstract

Although in utero hematopoietic cell transplantation is a promising strategy to treat congenital hematopoietic disorders, levels of engraftment have not been therapeutic for diseases in which donor cells have no survival advantage. We used an antibody against the murine c-Kit receptor (ACK2) to deplete fetal host hematopoietic stem cells (HSCs) and increase space within the hematopoietic niche for donor cell engraftment. Fetal mice were injected with ACK2 on embryonic days 13.5 to 14.5 and surviving pups were transplanted with congenic hematopoietic cells on day of life 1. Low-dose ACK2 treatment effectively depleted HSCs within the bone marrow with minimal toxicity and the antibody was cleared from the serum before the neonatal transplantation. Chimerism levels were significantly higher in treated pups than in controls; both myeloid and lymphoid cell chimerism increased because of higher engraftment of HSCs in the bone marrow. To test the strategy of repeated HSC depletion and transplantation, some mice were treated with ACK2 postnatally, but the increase in engraftment was lower than that seen with prenatal treatment. We demonstrate a successful fetal conditioning strategy associated with minimal toxicity. Such strategies could be used to achieve clinically relevant levels of engraftment to treat congenital stem cell disorders.

Abstract

The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

Abstract

Hematopoietic stem cells (HSCs) maintain homeostasis and regenerate the blood system throughout life. It has been postulated that HSCs may be uniquely capable of preserving their genomic integrity in order to ensure lifelong function. To directly test this, we quantified DNA damage in HSCs and downstream progenitors from young and old mice, revealing that strand breaks significantly accrue in HSCs during aging. DNA damage accumulation in HSCs was associated with broad attenuation of DNA repair and response pathways that was dependent upon HSC quiescence. Accordingly, cycling fetal HSCs and adult HSCs driven into cycle upregulated these pathways leading to repair of strand breaks. Our results demonstrate that HSCs are not comprehensively geno-protected during aging. Rather, HSC quiescence and concomitant attenuation of DNA repair and response pathways underlies DNA damage accumulation in HSCs during aging. These results provide a potential mechanism through which premalignant mutations accrue in HSCs.

Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.Proceedings of the National Academy of Sciences of the United States of AmericaAli, S. R., Hippenmeyer, S., Saadat, L. V., Luo, L., Weissman, I. L., Ardehali, R.2014; 111 (24): 8850-8855

Abstract

The mammalian heart has long been considered a postmitotic organ, implying that the total number of cardiomyocytes is set at birth. Analysis of cell division in the mammalian heart is complicated by cardiomyocyte binucleation shortly after birth, which makes it challenging to interpret traditional assays of cell turnover [Laflamme MA, Murray CE (2011) Nature 473(7347):326-335; Bergmann O, et al. (2009) Science 324(5923):98-102]. An elegant multi-isotope imaging-mass spectrometry technique recently calculated the low, discrete rate of cardiomyocyte generation in mice [Senyo SE, et al. (2013) Nature 493(7432):433-436], yet our cellular-level understanding of postnatal cardiomyogenesis remains limited. Herein, we provide a new line of evidence for the differentiated ?-myosin heavy chain-expressing cardiomyocyte as the cell of origin of postnatal cardiomyogenesis using the "mosaic analysis with double markers" mouse model. We show limited, life-long, symmetric division of cardiomyocytes as a rare event that is evident in utero but significantly diminishes after the first month of life in mice; daughter cardiomyocytes divide very seldom, which this study is the first to demonstrate, to our knowledge. Furthermore, ligation of the left anterior descending coronary artery, which causes a myocardial infarction in the mosaic analysis with double-marker mice, did not increase the rate of cardiomyocyte division above the basal level for up to 4 wk after the injury. The clonal analysis described here provides direct evidence of postnatal mammalian cardiomyogenesis.

Abstract

The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

Abstract

Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.

Abstract

Analysis of hematopoietic stem cell function in nonhuman primates provides insights that are relevant for human biology and therapeutic strategies. In this study, we applied quantitative genetic barcoding to track the clonal output of transplanted autologous rhesus macaque hematopoietic stem and progenitor cells over a time period of up to 9.5 months. We found that unilineage short-term progenitors reconstituted myeloid and lymphoid lineages at 1 month but were supplanted over time by multilineage clones, initially myeloid restricted, then myeloid-B clones, and then stable myeloid-B-T multilineage, long-term repopulating clones. Surprisingly, reconstitution of the natural killer (NK) cell lineage, and particularly the major CD16(+)/CD56(-) peripheral blood NK compartment, showed limited clonal overlap with T, B, or myeloid lineages, and therefore appears to be ontologically distinct. Thus, in addition to providing insights into clonal behavior over time, our analysis suggests an unexpected paradigm for the relationship between NK cells and other hematopoietic lineages in primates.

Abstract

Cancer is widely characterized by the sequential acquisition of genetic lesions in a single lineage of cells. Our previous studies have shown that, in acute myeloid leukemia (AML), mutation acquisition occurs in functionally normal hematopoietic stem cells (HSCs). These preleukemic HSCs harbor some, but not all, of the mutations found in the leukemic cells. We report here the identification of patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, whereas mutations in "proliferative" genes occur late. Additionally, we analyze the persistence of preleukemic mutations in patients in remission and find CD34+ progenitor cells and various mature cells that harbor preleukemic mutations. These findings indicate that preleukemic HSCs can survive induction chemotherapy, identifying these cells as a reservoir for the reevolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease and show that some of these patterns are consistent with involvement of preleukemic HSCs. These findings provide key insights into the monitoring of minimal residual disease and the identification of therapeutic targets in human AML.

Abstract

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

Abstract

Cationic, amphipathic host defense peptides represent a promising group of agents to be developed for anticancer applications. Poly-N-substituted glycines, or peptoids, are a class of biostable, peptidomimetic scaffold that can display a great diversity of side chains in highly tunable sequences via facile solid-phase synthesis. Herein, we present a library of anti-proliferative peptoids that mimics the cationic, amphipathic structural feature of the host defense peptides and explore the relationships between the structure, anticancer activity and selectivity of these peptoids. Several peptoids are found to be potent against a broad range of cancer cell lines at low-micromolar concentrations including cancer cells with multidrug resistance (MDR), causing cytotoxicity in a concentration-dependent manner. They can penetrate into cells, but their cytotoxicity primarily involves plasma membrane perturbations. Furthermore, peptoid 1, the most potent peptoid synthesized, significantly inhibited tumor growth in a human breast cancer xenotransplantation model without any noticeable acute adverse effects in mice. Taken together, our work provided important structural information for designing host defense peptides or their mimics for anticancer applications. Several cationic, amphipathic peptoids are very attractive for further development due to their high solubility, stability against protease degradation, their broad, potent cytotoxicity against cancer cells and their ability to overcome multidrug resistance.

Abstract

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease. As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation. An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Abstract

The use of C57BL/6 Rag2(-/-)?c(-/-) mice as recipients for xenotransplantation with human immune systems (humanization) has been problematic because C57BL/6 SIRP? does not recognize human CD47, and such recognition is required to suppress macrophage-mediated phagocytosis of transplanted human hematopoietic stem cells (HSCs). We show that genetic inactivation of CD47 on the C57BL/6 Rag2(-/-)?c(-/-) background negates the requirement for CD47-signal recognition protein ? (SIRP?) signaling and induces tolerance to transplanted human HSCs. These triple-knockout, bone marrow, liver, thymus (TKO-BLT) humanized mice develop organized lymphoid tissues including mesenteric lymph nodes, splenic follicles and gut-associated lymphoid tissue that demonstrate high levels of multilineage hematopoiesis. Importantly, these mice have an intact complement system and showed no signs of graft-versus-host disease (GVHD) out to 29 weeks after transplantation. Sustained, high-level HIV-1 infection was observed via either intrarectal or intraperitoneal inoculation. TKO-BLT mice exhibited hallmarks of human HIV infection including CD4(+) T-cell depletion, immune activation, and development of HIV-specific B- and T-cell responses. The lack of GVHD makes the TKO-BLT mouse a significantly improved model for long-term studies of pathogenesis, immune responses, therapeutics, and vaccines to human pathogens.

Abstract

Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of "liquid biopsies" from cancer patients.

Abstract

Human pluripotent stem cells (PSCs) are a leading candidate for cell-based therapies because of their capacity for unlimited self renewal and pluripotent differentiation. These advances have recently culminated in the first-in-human PSC clinical trials by Geron, Advanced Cell Technology and the Kobe Center for Developmental Biology for the treatment of spinal cord injury and macular degeneration. Despite their therapeutic promise, a crucial hurdle for the clinical implementation of human PSCs is their potential to form tumors in vivo. In this Perspective, we present an overview of the mechanisms underlying the tumorigenic risk of human PSC-based therapies and discuss current advances in addressing these challenges.

Abstract

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

Abstract

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

Abstract

CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRP?, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRP? variants with approximately 50,000-fold increased affinity for human CD47 relative to wild-type SIRP?. As high-affinity SIRP? monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.

Abstract

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.

Abstract

Glioblastoma multiforme (GBM) is the most common primary brain tumor, with a median survival of only 15 months. A subpopulation of cells, the brain tumor stem cells (BTSCs), may be responsible for the malignancy of this disease. Xenografts have proven to be a robust model of human BTSCs, but the effects of long-term passaging have yet to be determined. Here we present a study detailing changes in BTSC multipotency, invasive migration, and proliferation after serial passaging of human GBM xenografts. Immunocytochemistry and tumorsphere formation assays demonstrated the presence of BTSCs in both early generation (EG-BTSCs; less than 15 passages) and late generation (LG-BTSCs; more than 24 passages) xenografts. The EG-BTSCs upregulated expression of lineage markers for neurons and oligodendrocytes upon differentiation, indicating multipotency. In contrast, the LG-BTSCs were restricted to an astrocytic differentiation. Quantitative migration and proliferation assays showed that EG-BTSCs are more migratory and proliferative than LG-BTSCs. However, both populations respond similarly to the chemokine SDF-1 by increasing invasive migration. These differences between the EG- and LG-BTSCs were correlated with a significant decrease in nanog expression as determined by qRT-PCR. Mice implanted intracranially with EG-BTSCs showed shorter survival when compared to LG-BTSCs. Moreover, differentiation prior to implantation of EG-BTSCs, but not LG-BTSCs, led to increased survival. Thus, nanog may identify multipotent BTSCs. Furthermore, limited passaging of xenografts preserves these multipotent BTSCs, which may be an essential underlying feature of GBM lethality.

Abstract

Epigenetic therapies demonstrate significant clinical activity in acute myeloid leukemia (AML) and myelodysplasia (MDS) and constitute an important new class of therapeutic agents. However hematological responses are not durable and disease relapse appears inevitable. Experimentally, leukemic stem/progenitor cells (LSC) propagate disease in animal models of AML and it has been postulated that their relative chemo-resistance contributes to disease relapse. We serially measured LSC numbers in patients with high-risk AML and MDS treated with 5'-azacitidine and sodium valproate (VAL-AZA). Fifteen out of seventy-nine patients achieved a complete remission (CR) or complete remission with incomplete blood count recovery (CRi) with VAL-AZA therapy. There was no significant reduction in the size of the LSC-containing population in non-responders. While the LSC-containing population was substantially reduced in all patients achieving a CR/CRi it was never eradicated and expansion of this population antedated morphological relapse. Similar studies were performed in seven patients with newly diagnosed AML treated with induction chemotherapy. Eradication of the LSC-containing population was observed in three patients all of whom achieved a durable CR in contrast to patients with resistant disease where LSC persistence was observed. LSC quantitation provides a novel biomarker of disease response and relapse in patients with AML treated with epigenetic therapies. New drugs that target this cellular population in vivo are required.

Abstract

Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70-80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70-85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.

Abstract

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFR?(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFR? in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFR?(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

Abstract

Myelodysplastic syndromes (MDS) are a group of disorders characterized by variable cytopenias and ineffective hematopoiesis. Hematopoietic stem cells (HSCs) and myeloid progenitors in MDS have not been extensively characterized. We transplanted purified human HSCs from MDS samples into immunodeficient mice and show that HSCs are the disease-initiating cells in MDS. We identify a recurrent loss of granulocyte-macrophage progenitors (GMPs) in the bone marrow of low risk MDS patients that can distinguish low risk MDS from clinical mimics, thus providing a simple diagnostic tool. The loss of GMPs is likely due to increased apoptosis and increased phagocytosis, the latter due to the up-regulation of cell surface calreticulin, a prophagocytic marker. Blocking calreticulin on low risk MDS myeloid progenitors rescues them from phagocytosis in vitro. However, in the high-risk refractory anemia with excess blasts (RAEB) stages of MDS, the GMP population is increased in frequency compared with normal, and myeloid progenitors evade phagocytosis due to up-regulation of CD47, an antiphagocytic marker. Blocking CD47 leads to the selective phagocytosis of this population. We propose that MDS HSCs compete with normal HSCs in the patients by increasing their frequency at the expense of normal hematopoiesis, that the loss of MDS myeloid progenitors by programmed cell death and programmed cell removal are, in part, responsible for the cytopenias, and that up-regulation of the "don't eat me" signal CD47 on MDS myeloid progenitors is an important transition step leading from low risk MDS to high risk MDS and, possibly, to acute myeloid leukemia.

Abstract

The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life.

Abstract

The potential to develop into any cell type makes human pluripotent stem cells (hPSCs) one of the most promising sources for regenerative treatments. Hurdles to their clinical applications include (1) formation of heterogeneously differentiated cultures, (2) the risk of teratoma formation from residual undifferentiated cells, and (3) immune rejection of engrafted cells. The recent production of human isogenic (genetically identical) induced PSCs (hiPSCs) has been proposed as a "solution" to the histocompatibility barrier. In theory, differentiated cells derived from patient-specific hiPSC lines should be histocompatible to their donor/recipient. However, propagation, maintenance, and non-physiologic differentiation of hPSCs in vitro may produce other, likely less powerful, immune responses. In light of recent progress towards the clinical application of hPSCs, this review focuses on two antigen presentation phenomena that may lead to rejection of isogenic hPSC derivates: namely, the expression of aberrant antigens as a result of long-term in vitro maintenance conditions or incomplete somatic cell reprogramming, and the unbalanced presentation of receptors and ligands involved in immune recognition due to accelerated differentiation. Finally, we discuss immunosuppressive approaches that could potentially address these immunological concerns.

Abstract

Acquired resistance to imatinib is a significant problem for the clinical management of gastrointestinal stromal tumor (GIST) patients, and second-line small molecules have shown limited efficacy in this setting. We have recently demonstrated that a monoclonal antibody targeting KIT could potentially bypass imatinib resistance in preclinical models of GIST.

Abstract

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.

Abstract

Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot find VSELs in mouse BM with any of the reported stem cell potentials, specifically for hematopoiesis. We found that: (1) most events within the "VSEL" flow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to confirm the existence of pluripotent VSELs.

Abstract

Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

Abstract

Fibroblasts and smooth muscle cells (FSMCs) are principal cell types of connective and adventitial tissues that participate in the development, physiology and pathology of internal organs, with incompletely defined cellular origins. Here, we identify and prospectively isolate from the mesothelium a mouse cell lineage that is committed to FSMCs. The mesothelium is an epithelial monolayer covering the vertebrate thoracic and abdominal cavities and internal organs. Time-lapse imaging and transplantation experiments reveal robust generation of FSMCs from the mesothelium. By targeting mesothelin (MSLN), a surface marker expressed on mesothelial cells, we identify and isolate precursors capable of clonally generating FSMCs. Using a genetic lineage tracing approach, we show that embryonic and adult mesothelium represents a common lineage to trunk FSMCs, and trunk vasculature, with minimal contributions from neural crest, or circulating cells. The isolation of FSMC precursors enables the examination of multiple aspects of smooth muscle and fibroblast biology as well as the prospective isolation of these precursors for potential regenerative medicine purposes.

Abstract

Multiple myeloma is a hematological neoplasm characterized by the accumulation of clonal plasma cells in the bone marrow. Its frequent relapse following achievement of clinical remissions implicates the existence of therapy-resistant myeloma-initiating cells. To date, results on the identity of myeloma-initiating cells have differed. Here, we prospectively identified a myeloma-initiating population by fractionating and transplanting patient bone marrow cells into human bone-bearing immunocompromised mice. Xenotransplantation of fractionated CD138(+)/CD38(high) cells from 40% of patients (8/20) led to a repopulation of CD19(+)CD38(low) or CD138(+)CD38(+) B-lineage cells in human bone grafts; and these grafts were clonally derived from patient myeloma cells. Meanwhile, CD19(+)CD38(low) xenografts were detected in human bone-bearing mice transplanted with CD19(+)CD38(low/-) B cells from 8 of 22 samples but were not clonally related to patient myeloma cells. Further fractionation and xenotransplantation of CD138(+)CD38(high) cells demonstrated that (CD45(low/-) or CD19(-)) CD38(high)/CD138(+) plasma cells, but not (CD45(high) or CD19(+)) CD38(high)/CD138(+) plasmablasts enrich for myeloma-initiating cells. Quantitative reverse transcription-PCR of two serially transplantable xenografts, which were CD19(-)CD138(+), revealed that they were Pax5 (a B-cell-specific transactivator)-negative. These results suggest that CD19(-)CD45(low/-) fully differentiated plasma cells enrich for long-lived and tumor-initiating cells whereas B cells or plasmablasts do not.

Abstract

Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.

Abstract

Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC.

Abstract

Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.

Abstract

Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

Abstract

Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein ? (SIRP-?). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

Abstract

Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1-specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell-based therapy approaches.

Abstract

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRP?, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.

Abstract

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-?, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

Abstract

Multiple lines of investigation have demonstrated that the immune system plays an important role in preventing tumor initiation and controlling tumor growth. Accordingly, many cancers have evolved diverse mechanisms to evade such monitoring. While multiple immune cell types mediate tumor surveillance, recent evidence demonstrates that macrophages, and other phagocytic cells, play a key role in regulating tumor growth through phagocytic clearance. In this review we highlight the role of tumor immune evasion through the inhibition of phagocytosis, specifically through the CD47-signal-regulatory protein-? pathway, and discuss how targeting this pathway might lead to more effective cancer immunotherapies.

Abstract

Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc(-/-) embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc(-/-) embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc(-/-) embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc(-/-) embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc(-/-) embryos is not the result of intrinsic HSC failure.

Abstract

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

Abstract

The development of cancer involves mechanisms by which aberrant cells overcome normal regulatory pathways that limit their numbers and their migration. The evasion of programmed cell death is one of several key early events that need to be overcome in the progression from normal cellular homeostasis to malignant transformation. Recently, we provided evidence in mouse and human cancers that successful cancer clones must also overcome programmed cell removal. In this Opinion article, we explore the role of programmed cell removal in both normal and neoplastic cells, and we place this pathway in the context of the initiation of programmed cell death.

Abstract

We previously identified by flow cytometry a Lineage-CD44+ (Lin-CD44+) subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma (HNSCC). We now correlate clinical and histologic factors with Lin-CD44+ cell frequency.The study included 31 patients with HNSCC, of whom 87% had stage IV disease. The frequency of Lin-CD44+ cells and the success of xenografting patient tumors in mice were correlated with clinical and pathologic data.The mean frequency of Lin-CD44+ cells was 25% (0.4%-81%). It was 36% in patients who had recurrence versus 15% for those without recurrence (p = .04). Successful xenograft implantation occurred in 53%. Seventy-five percent of patients with successful xenografts had recurrence versus 21% of patients with unsuccessful xenografts (p = .003).Successful xenograft implantation and a high frequency of Lin-CD44+ cells correlate with known poor prognostic factors such as advanced T classification and recurrence. These findings may support the stem cell concept in HNSCC.

Abstract

Metastatic breast cancer remains a major treatment challenge. The use of high-dose chemotherapy (HDCT) with rescue by autologous mobilized peripheral blood (MPB) is controversial, in part because of contamination of MPB by circulating tumor cells. CD34(+)Thy-1(+) selected hematopoietic stem cells (HSC) represent a graft source with a greater than 250,000-fold reduction in cancer cells. Here, we present the long-term outcome of a pilot study to determine feasibility and engraftment using HDCT and purified HSC in patients with metastatic breast cancer. Twenty-two patients who had been treated with standard chemotherapy were enrolled into a phase I/II trial between December 1996 and February 1998, and underwent HDCT followed by rescue with CD34(+)Thy-1(+) HSC isolated from autologous MPB. More than 12 years after the end of the study, 23% (5 of 22) of HSC recipients are alive, and 18% (4 of 22) are free of recurrence with normal hematopoietic function. Median progression-free survival (PFS) was 16 months, and median overall survival (OS) was 60 months. Retrospective comparison with 74 patients transplanted between February 1995 and June 1999 with the identical HDCT regimen but rescue with unmanipulated MPB indicated that 9% of patients are alive, and 7% are without disease. Median PFS was 10 months, and median OS was 28 months. In conclusion, cancer-depleted HSC following HDCT resulted in better than expected 12- to 14-year PFS and OS in a cohort of metastatic breast cancer patients. These data prompt us to look once again at purified HSC transplantation in a protocol powered to test for efficacy in advanced-stage breast cancer patients.

Abstract

In the human hematopoietic system, aging is associated with decreased bone marrow cellularity, decreased adaptive immune system function, and increased incidence of anemia and other hematological disorders and malignancies. Recent studies in mice suggest that changes within the hematopoietic stem cell (HSC) population during aging contribute significantly to the manifestation of these age-associated hematopoietic pathologies. Though the mouse HSC population has been shown to change both quantitatively and functionally with age, changes in the human HSC and progenitor cell populations during aging have been incompletely characterized. To elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated immunophenotypic HSC and other hematopoietic progenitor populations from healthy, hematologically normal young and elderly human bone marrow samples. We found that aged immunophenotypic human HSC increase in frequency, are less quiescent, and exhibit myeloid-biased differentiation potential compared with young HSC. Gene expression profiling revealed that aged immunophenotypic human HSC transcriptionally up-regulate genes associated with cell cycle, myeloid lineage specification, and myeloid malignancies. These age-associated alterations in the frequency, developmental potential, and gene expression profile of human HSC are similar to those changes observed in mouse HSC, suggesting that hematopoietic aging is an evolutionarily conserved process.

Abstract

Natural killer (NK) cells develop in the bone marrow and are known to gradually acquire the ability to eliminate infected and malignant cells, yet the cellular stages of NK lineage commitment and maturation are incompletely understood. Using 12-color flow cytometry, we identified a novel NK-committed progenitor (pre-NKP) that is a developmental intermediate between the upstream common lymphoid progenitor and the downstream NKP, previously assumed to represent the first stage of NK lineage commitment. Our analysis also refined the purity of NKPs (rNKP) by 6-fold such that 50% of both pre-NKP and rNKP cells gave rise to NKp46+ NK cells at the single-cell level. On transplantation into unconditioned Rag2-/-Il2r?c-/- recipients, both pre-NKPs and rNKPs generated mature NK cells expressing a repertoire of Ly49 family members that degranulated on stimulation ex vivo. Intrathymic injection of these progenitors, however, yielded no NK cells, suggesting a separate origin of thymic NK cells. Unlike the rNKP, the pre-NKP does not express IL-2R? (CD122), yet it is lineage committed toward the NK cell fate, adding support to the theory that IL-15 signaling is not required for NK commitment. Taken together, our data provide a high-resolution in vivo analysis of the earliest steps of NK cell commitment and maturation.

Abstract

Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibody-mediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRP? and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors.

Abstract

Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long-term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO-conjugated monoclonal antibody against these antigens (SPIO-MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO-MAb. The human and mouse ESCs were transduced with the RG (ESC-RGs) and transplanted into the peri-infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO-MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC-derived teratoma formation in the injured murine myocardium.

Abstract

Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self-renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (-) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed "FSE cells." GATA1-low progenitors were identified as KLS Flk2(+), CD150(-), and cMPL(-), and were termed "Flk(+) CD150(-) cells." FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony-forming unit-spleen activity compared with Flk(+) CD150(-) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term HSCs and FSE cells, but not in Flk(+) CD150(-) cells in response to stimulation with thrombopoietin, an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.

Abstract

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.

Abstract

Reduced gene dosage of ribosomal protein subunits has been implicated in 5q- myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q- syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.

Abstract

The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

Abstract

The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.

Abstract

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

Abstract

The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials.

Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factorsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAArdehali, R., Inlay, M. A., Ali, S. R., Tang, C., Drukker, M., Weissman, I. L.2011; 108 (8): 3282-3287

Abstract

The promise of pluripotent stem cells as a research and therapeutic tool is partly undermined by the technical challenges of generating and maintaining these cells in culture. Human embryonic stem cells (hESCs) are exquisitely sensitive to culture conditions, and require constant signaling by growth factors and cell-cell and cell-matrix interactions to prevent apoptosis, senescence, and differentiation. Previous work from our laboratory demonstrated that overexpression of the prosurvival gene BCL2 in mouse embryonic stem cells overrode the requirement of serum factors and feeder cells to maintain mESCs in culture. To determine whether this prosurvival gene could similarly protect hESCs, we generated hESC lines that constitutively or inducibly express BCL2. We find that BCL2 overexpression significantly decreases dissociation-induced apoptosis, resulting in enhanced colony formation from sorted single cells, and enhanced embryoid body formation. In addition, BCL2-hESCs exhibit normal growth in the absence of serum, but require basic fibroblast growth factor to remain undifferentiated. Furthermore, they maintain their pluripotency markers, form teratomas in vivo, and differentiate into all three germ layers. Our data suggest that the BCL2 signaling pathway plays an important role in inhibiting hESC apoptosis, such that its overexpression in hESCs offers both a survival benefit in conditions of stress by resisting apoptosis and obviates the requirement for serum or a feeder layer for maintenance.

Abstract

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and constitutes 15% of adult leukemias. Although overall prognosis for pediatric ALL is favorable, high-risk pediatric patients and most adult patients have significantly worse outcomes. Multiagent chemotherapy is standard of care for both pediatric and adult ALL, but is associated with systemic toxicity and long-term side effects and is relatively ineffective against certain ALL subtypes. Recent efforts have focused on the development of targeted therapies for ALL including monoclonal antibodies. Here, we report the identification of CD47, a protein that inhibits phagocytosis, as an antibody target in standard and high-risk ALL. CD47 was found to be more highly expressed on a subset of human ALL patient samples compared with normal cell counterparts and to be an independent predictor of survival and disease refractoriness in several ALL patient cohorts. In addition, a blocking monoclonal antibody against CD47 enabled phagocytosis of ALL cells by macrophages in vitro and inhibited tumor engraftment in vivo. Significantly, anti-CD47 antibody eliminated ALL in the peripheral blood, bone marrow, spleen, and liver of mice engrafted with primary human ALL. These data provide preclinical support for the development of an anti-CD47 antibody therapy for treatment of human ALL.

Abstract

Replacement of disease-causing stem cells with healthy ones has been achieved clinically via hematopoietic cell transplantation (HCT) for the last 40 years, as a treatment modality for a variety of cancers and immunodeficiencies with moderate, but increasing, success. This procedure has traditionally included transplantation of mixed hematopoietic populations that include hematopoietic stem cells (HSC) and other cells, such as T cells. This article explores and delineates the potential expansion of this technique to treat a variety of inherited diseases of immune function, the current barriers in HCT and pure HSC transplantation, and the up-and-coming strategies to combat these obstacles.

Abstract

Transcription factor NF-?B and its activating kinase IKK? are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKK?-NF-?B has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKK? (Ikk?(?) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikk?(?) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikk?(?)Il1r1(-/-) mice, unlike Ikk?(?) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKK?-NF-?B or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

Abstract

A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKC?, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKC?, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKC? leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

Abstract

In this issue of Cancer Cell, Goardon et al. revise earlier conclusions regarding acute myelogenous leukemia (AML) stem cells by demonstrating that in the majority of patients, they reside in two hierarchically related populations most similar to normal hematopoietic progenitors. These findings have implications for therapeutic targeting of these cells.

Abstract

Under normal physiological conditions, cellular homeostasis is partly regulated by a balance of pro- and anti-phagocytic signals. CD47, which prevents cancer cell phagocytosis by the innate immune system, is highly expressed on several human cancers including acute myeloid leukemia, non-Hodgkin's lymphoma, and bladder cancer. Blocking CD47 with a monoclonal antibody results in phagocytosis of cancer cells and leads to in vivo tumor elimination, yet normal cells remain mostly unaffected. Thus, we postulated that cancer cells must also display a potent pro-phagocytic signal. Here, we identified calreticulin as a pro-phagocytic signal that was highly expressed on the surface of several human cancers, but was minimally expressed on most normal cells. Increased CD47 expression correlated with high amounts of calreticulin on cancer cells and was necessary for protection from calreticulin-mediated phagocytosis. Blocking the interaction of target cell calreticulin with its receptor, low-density lipoprotein receptor-related protein, on phagocytic cells prevented anti-CD47 antibody-mediated phagocytosis. Furthermore, increased calreticulin expression was an adverse prognostic factor in diverse tumors including neuroblastoma, bladder cancer, and non-Hodgkin's lymphoma. These findings identify calreticulin as the dominant pro-phagocytic signal on several human cancers, provide an explanation for the selective targeting of tumor cells by anti-CD47 antibody, and highlight the balance between pro- and anti-phagocytic signals in the immune evasion of cancer.

MicroRNA-125b expands hematopoietic stem cells and enriches for the lymphoid-balanced and lymphoid-biased subsetsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAOoi, A. G., Sahoo, D., Adorno, M., Wang, Y., Weissman, I. L., Park, C. Y.2010; 107 (50): 21505-21510

Abstract

MicroRNAs profoundly impact hematopoietic cells by regulating progenitor cell-fate decisions, as well as mature immune effector function. However to date, microRNAs that regulate hematopoietic stem cell (HSC) function have been less well characterized. Here we show that microRNA-125b (miR-125b) is highly expressed in HSCs and its expression decreases in committed progenitors. Overexpression of miR-125b in mouse HSC enhances their function, demonstrated through serial transplantation of highly purified HSC, and enriches for the previously described Slamf1(lo)CD34(-) lymphoid-balanced and the Slamf1(neg)CD34(-) lymphoid-biased cell subsets within the multipotent HSC (CD34-KLS) fraction. Mature peripheral blood cells derived from the miR-125b-overexpressing HSC are skewed toward the lymphoid lineage. Consistent with this observation, miR-125b overexpression significantly increases the number of early B-progenitor cells within the spleen and induces the expansion and enrichment of the lymphoid-balanced and lymphoid-biased HSC subset via an antiapoptotic mechanism, reducing the mRNA expression levels of two proapoptotic targets, Bmf and KLF13. The antiapoptotic effect of miR-125b is more pronounced in the lymphoid-biased HSC subset because of their intrinsic higher baseline levels of apoptosis. These effects of miR-125b are associated with the development of lymphoproliferative disease, marked by expansion of CD8(+) T lymphocytes. Taken together, these data reveal that miR-125b regulates HSC survival and can promote lymphoid-fate decisions at the level of the HSC by preferentially expanding lymphoid-balanced and lymphoid-biased HSC.

T-cell receptor-driven lymphomagenesis in mice derived from a reprogrammed T cellPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASerwold, T., Hochedlinger, K., Swindle, J., Hedgpeth, J., Jaenisch, R., Weissman, I. L.2010; 107 (44): 18939-18943

Abstract

The conversion of mature somatic cells into pluripotent stem cells, both by nuclear transfer and transduction with specific "reprogramming" genes, represents a major advance in regenerative medicine. Pluripotent stem cell lines can now be generated from an individual's own cells, facilitating the generation of immunologically acceptable stem cell-based therapeutics. Many cell types can undergo nuclear reprogramming, leading to the question of whether the identity of the reprogrammed cell of origin has a biological consequence. Peripheral blood, containing a mixture of T, B, NK, and myeloid cell types, represents one potential source of reprogrammable cells. In this study, we describe the unique case of mice derived from a reprogrammed T cell. These mice have prerearranged T-cell receptor (TCR) genes in all cells. Surprisingly, ?50% of mice with prerearranged TCR genes develop spontaneous T cell lymphomas, which originate in the thymus. The lymphomas arise from developing T cells, and contain activated Notch1, similar to most human and mouse T-cell acute lymphoblastic lymphomas. Furthermore, lymphomagenesis requires the expression of both prerearranged TCR? and TCR? genes, indicating a critical role for TCR signaling. Furthermore, inhibitors of multiple branches of TCR signaling suppress lymphoma growth, implicating TCR signaling as an essential component in lymphoma proliferation. The lymphomagenesis in mice derived from a reprogrammed T cell demonstrates the deleterious consequences of misregulation of the TCR rearrangement and signaling pathways and illustrates one case of cellular reprogramming where the identity of the cell of origin has profound consequences.

Abstract

The mammalian blood system, containing more than 10 distinct mature cell types, stands on one specific cell type, hematopoietic stem cell (HSC). Within the system, only HSCs possess the ability of both multipotency and self-renewal. Multipotency is the ability to differentiate into all functional blood cells. Self-renewal is the ability to give rise to HSC itself without differentiation. Since mature blood cells (MBCs) are predominantly short-lived, HSCs continuously provide more differentiated progenitors while properly maintaining the HSC pool size throughout life by precisely balancing self-renewal and differentiation. Thus, understanding the mechanisms of self-renewal and differentiation of HSC has been a central issue. In this review, we focus on the hierarchical structure of the hematopoietic system, the current understanding of microenvironment and molecular cues regulating self-renewal and differentiation of adult HSCs, and the currently emerging systems approaches to understand HSC biology.

Abstract

Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

Abstract

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

Abstract

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Abstract

The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

Abstract

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

Abstract

Recently, the prospective isolation and characterization of cancer stem cells (CSCs) from various human malignancies revealed that they are resistant to radiation and chemotherapies. Therefore, CSCs may be the 'roots' and ideal target for therapeutic intervention. Here, we will focus on reviewing the historical perspective, recent literatures on bladder cancer stem cells and their clinical implications.CSCs have been prospectively isolated from bladder cancer tissues from patient specimens, established cancer cell lines and xenografts, based on the expression of a combination of cell surface receptors, cytokeratin markers, drug transporters and the efficient efflux of the Hoechst 33,342 dye (side population). Further, global gene expression profiling of CSCs revealed an activated gene signature of CSCs similar to that of aggressive bladder cancer, supporting the concept that a tumor cell subpopulation is contributing to bladder cancer progression. Finally, our studies on the preclinical targeting of bladder CSCs in vitro and in xenografts using a blocking antibody for CD47 reveal promising efficacy.Functionally distinct CSCs exist in human bladder cancer and can be prospectively isolated. Continuing research will be important to identify their cell of origin, programs balancing self-renewal and differentiation and to identify additional therapeutic options to target bladder CSCs.

Abstract

The long lifespan of tissue-specific stem cells suggests that they may respond differently to DNA damage than downstream cells. In this issue of Cell Stem Cell, two groups address this hypothesis by examining DNA damage responses in hematopoietic stem and progenitor cells (Milyavsky et al., 2010; Mohrin et al., 2010).

Abstract

A report by the International Society for Stem Cell Research (ISSCR)'s Task Force on Unproven Stem Cell Treatments outlines development of resources for patients, their families, and physicians seeking information on stem cell treatments.

Abstract

The question of whether tumorigenic cancer stem cells exist in human melanomas has arisen in the last few years. Here we show that in melanomas, tumour stem cells (MTSCs, for melanoma tumour stem cells) can be isolated prospectively as a highly enriched CD271(+) MTSC population using a process that maximizes viable cell transplantation. The tumours sampled in this study were taken from a broad spectrum of sites and stages. High-viability cells isolated by fluorescence-activated cell sorting and re-suspended in a matrigel vehicle were implanted into T-, B- and natural-killer-deficient Rag2(-/-)gammac(-/-) mice. The CD271(+) subset of cells was the tumour-initiating population in 90% (nine out of ten) of melanomas tested. Transplantation of isolated CD271(+) melanoma cells into engrafted human skin or bone in Rag2(-/-)gammac(-/-) mice resulted in melanoma; however, melanoma did not develop after transplantation of isolated CD271(-) cells. We also show that in mice, tumours derived from transplanted human CD271(+) melanoma cells were capable of metastatsis in vivo. CD271(+) melanoma cells lacked expression of TYR, MART1 and MAGE in 86%, 69% and 68% of melanoma patients, respectively, which helps to explain why T-cell therapies directed at these antigens usually result in only temporary tumour shrinkage.

Abstract

Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPalpha upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPalpha interaction.

Abstract

Replacement of disease-causing stem cells with healthy ones has been achieved clinically via hematopoietic cell transplantation (HCT) for the last 40 years, as a treatment modality for a variety of cancers and immunodeficiencies with moderate, but increasing, success. This procedure has traditionally included transplantation of mixed hematopoietic populations that include hematopoietic stem cells (HSC) and other cells, such as T cells. This article explores and delineates the potential expansion of this technique to treat a variety of inherited diseases of immune function, the current barriers in HCT and pure HSC transplantation, and the up-and-coming strategies to combat these obstacles.

Abstract

The lineage restriction of prospectively isolated hematopoietic progenitors has been traditionally assessed by bulk in vitro culture and transplantation of large number of cells in vivo. These methods, however, cannot distinguish between homogenous multipotent or heterogeneous lineage-restricted populations. Using clonal assays of 1 or 5 cells in vitro, single-cell quantitative gene expression analyses, and transplantation of mice with low numbers of cells, we show that a common myeloid progenitor (CMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(-) (SL-CMP; Sca-1(lo) CMP) and a granulocyte/macrophage progenitor (GMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(+)CD150(-/lo) (SL-GMP; Sca-1(lo) GMP). We found that mast cell progenitor potential is present in the SL-CMP fraction, but not in the more differentiated SL-GMP population, and is more closely related to megakaryocyte/erythrocyte specification. Our data provide criteria for the prospective isolation of SL-CMP and SL-GMP and support the conclusion that mast cells are specified during hematopoiesis earlier than and independently from granulocytes.

Abstract

We present a method termed mining developmentally regulated genes (MiDReG) to predict genes whose expression is either activated or repressed as precursor cells differentiate. MiDReG does not require gene expression data from intermediate stages of development. MiDReG is based on the gene expression patterns between the initial and terminal stages of the differentiation pathway, coupled with "if-then" rules (Boolean implications) mined from large-scale microarray databases. MiDReG uses two gene expression-based seed conditions that mark the initial and the terminal stages of a given differentiation pathway and combines the statistically inferred Boolean implications from these seed conditions to identify the relevant genes. The method was validated by applying it to B-cell development. The algorithm predicted 62 genes that are expressed after the KIT+ progenitor cell stage and remain expressed through CD19+ and AICDA+ germinal center B cells. qRT-PCR of 14 of these genes on sorted B-cell progenitors confirmed that the expression of 10 genes is indeed stably established during B-cell differentiation. Review of the published literature of knockout mice revealed that of the predicted genes, 63.4% have defects in B-cell differentiation and function and 22% have a role in the B cell according to other experiments, and the remaining 14.6% are not characterized. Therefore, our method identified novel gene candidates for future examination of their role in B-cell development. These data demonstrate the power of MiDReG in predicting functionally important intermediate genes in a given developmental pathway that is defined by a mutually exclusive gene expression pattern.

Abstract

Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus-the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart.

Abstract

Aging of the hematopoietic stem cell compartment is believed to contribute to the onset of a variety of age-dependent blood cell pathophysiologies. Mechanistic drivers of hematopoietic stem cell (HSC) aging include DNA damage accumulation and induction of tumor suppressor pathways that combine to reduce the regenerative capacity of aged HSCs. Such mechanisms do not however account for the change in lymphoid and myeloid lineage potential characteristic of HSC aging, which is believed to be central to the decline of immune competence and predisposition to myelogenous diseases in the elderly. Here we have prospectively isolated functionally distinct HSC clonal subtypes, based on cell surface phenotype, bearing intrinsically different capacities to differentiate toward lymphoid and myeloid effector cells mediated by quantitative differences in lineage priming. Finally, we present data supporting a model in which clonal expansion of a class of intrinsically myeloid-biased HSCs with robust self-renewal potential is a central component of hematopoietic aging.

Abstract

Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells.

Abstract

During mammalian development, as exemplified by mice, hematopoietic cells first appear in the yolk sac blood islands, then in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region and the placenta, eventually seeding into liver, spleen and then bone marrow. The formation of hematopoietic stem cells from mesodermal precursors has finished by mid-fetal life. Once established, the hematopoietic system must supply blood cells to host circulation and tissues for the entire life of the animal. Easy access to hematopoietic cells has enabled a vast number of studies over the last several decades, and much is now understood about the different hematopoietic lineages, how they differentiate, and their derivation from immature progenitors. Yet to be elucidated are the following two intriguing questions: do yolk sac and AGM hematopoietic cells arise from a common precursor or from distinct precursor cells?; and what is the lineage relationship between blood and endothelial cells. In this review, we will survey the state of our current knowledge in these areas, and discuss the potential use of multicolor chimera analyses to elucidate unresolved questions.

Abstract

Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time.In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods.Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future.

Abstract

Hematopoietic stem cells (HSCs) are rare, ancestral cells that underlie the development, homeostasis, aging, and regeneration of the blood. Here we show that the chromatin-associated protein Ikaros is a crucial self-renewal regulator of the long-term (LT) reconstituting subset of HSCs. Ikaros, and associated family member proteins, are highly expressed in self-renewing populations of stem cells. Ikaros point mutant mice initially develop LT-HSCs with the surface phenotype cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2-CD150+ during fetal ontogeny but are unable to maintain this pool, rapidly losing it within two days of embryonic development. A synchronous loss of megakaryocyte/erythrocyte progenitors results, along with a fatal, fetal anemia. At this time, mutation of Ikaros exerts a differentiation defect upon common lymphoid progenitors that cannot be rescued with an ectopic Notch signal in vitro, with hematopoietic cells preferentially committing to the NK lineage. Althoughdispensable for the initial embryonic development of blood, Ikaros is clearly needed for maintenance of this tissue. Achieving successful clinical tissue regeneration necessitates understanding degeneration, and these data provide a striking example by a discrete genetic lesion in the cells underpinning tissue integrity during a pivotal timeframe of organogenesis.

Abstract

Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that approximately 1-5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.

Abstract

Common lymphoid progenitors (CLPs) clonally produce both B- and T-cell lineages, but have little myeloid potential in vivo. However, some studies claim that the upstream lymphoid-primed multipotent progenitor (LMPP) is the thymic seeding population, and suggest that CLPs are primarily B-cell-restricted. To identify surface proteins that distinguish functional CLPs from B-cell progenitors, we used a new computational method of Mining Developmentally Regulated Genes (MiDReG). We identified Ly6d, which divides CLPs into two distinct populations: one that retains full in vivo lymphoid potential and produces more thymocytes at early timepoints than LMPP, and another that behaves essentially as a B-cell progenitor.

Abstract

Blood is a tissue with a high cell turnover rate that is constantly being replenished by bone marrow hematopoietic stem cells (HSCs) seeded during fetal ontogeny from the liver. Here we show that the long-term (LT) reconstituting subset of cKit(+)Thy1.1(lo)Lin(-/lo)Sca1(+)Flk2(-) HSCs is CD150(+). HSCs sourced from the fetal liver show LT, multilineage engraftment from E14.5 onward, and the CD150 cell surface molecule can readily substitute Thy1.1 as a positive marker of LT-HSCs in this tissue. From both fetal liver and adult bone marrow, cKit(+)Thy1.1(lo)Lin(-/lo)Sca1(+)Flk2(-) CD150(+) cells exhibit robust LT competitive engraftment, self-renewal, multilineage differentiation capacity, and an accessible chromatin configuration consistent with high expression of erythroid/megakaryoid genes in purified cell subsets. Our data show that, with appropriate combinations of cell surface markers, stem cells can be accurately isolated to high purity and characterized. This is important for the clarification of lineage relationships and the identification of bona fide regulators of stem cell self-renewal and differentiation both in normal and neoplastic tissues.

Abstract

This study investigated the ability of MnCl(2) as a cellular MRI contrast agent to determine the in vitro viability of human embryonic stem cells (hESC) and human bone marrow stromal cells (hBMSC). Basic MRI parameters including T(1) and T(2) values of MnCl(2)-labeled hESC and hBMSC were measured and viability signal of manganese (Mn(2+))-labeled cells was validated. Furthermore, the biological activity of Ca(2+)-channels was modulated utilizing both Ca(2+)-channel agonist and antagonist to evaluate concomitant signal changes. Metabolic effects of MnCl(2)-labeling were also assessed using assays for cell viability, proliferation, and apoptosis. Finally, in vivo Mn(2+)-guided MRI of the transplanted hESC was successfully achieved and validated by bioluminescence imaging.

Abstract

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1(-/)(-) mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology. Grafted mice displayed reduced autofluorescent lipofuscin, significant neuroprotection of host hippocampal and cortical neurons, and delayed loss of motor coordination. Early intervention with cellular transplants of hCNS-SCns into the brains of INCL patients may supply a continuous and long-lasting source of the missing PPT1 and provide some therapeutic benefit through protection of endogenous neurons. These data provide the experimental basis for human clinical trials with these banked hCNS-SCns.

Abstract

Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44(+)CK5(+)CK20(-)). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% beta-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

Abstract

Stem cells and their potential applications have become the forefront of scientific, political, and ethical discourse. Whereas stem cells were long accepted as units of development and evolution, it is now becoming increasingly clear that they are also units of oncogenesis. Although the field of stem cell biology is expanding at an astounding rate, the data attained are not readily translatable for the physicians who may eventually deliver these tools to patients. Herein, we provide a brief review of stem cell and cancer stem cell biology and highlight the scientific and clinical implications of recent findings regarding the presence of cancer-forming stem cells in brain tumors.

Abstract

Acute myeloid leukemia (AML) is organized as a cellular hierarchy initiated and maintained by a subset of self-renewing leukemia stem cells (LSC). We hypothesized that increased CD47 expression on human AML LSC contributes to pathogenesis by inhibiting their phagocytosis through the interaction of CD47 with an inhibitory receptor on phagocytes. We found that CD47 was more highly expressed on AML LSC than their normal counterparts, and that increased CD47 expression predicted worse overall survival in three independent cohorts of adult AML patients. Furthermore, blocking monoclonal antibodies directed against CD47 preferentially enabled phagocytosis of AML LSC and inhibited their engraftment in vivo. Finally, treatment of human AML LSC-engrafted mice with anti-CD47 antibody depleted AML and targeted AML LSC. In summary, increased CD47 expression is an independent, poor prognostic factor that can be targeted on human AML stem cells with blocking monoclonal antibodies capable of enabling phagocytosis of LSC.

Abstract

Macrophages clear pathogens and damaged or aged cells from the blood stream via phagocytosis. Cell-surface CD47 interacts with its receptor on macrophages, SIRPalpha, to inhibit phagocytosis of normal, healthy cells. We find that mobilizing cytokines and inflammatory stimuli cause CD47 to be transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to and during their migratory phase, and that the level of CD47 on these cells determines the probability that they are engulfed in vivo. CD47 is also constitutively upregulated on mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis. We conclude that CD47 upregulation is an important mechanism that provides protection to normal HSCs during inflammation-mediated mobilization, and that leukemic progenitors co-opt this ability in order to evade macrophage killing.

Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse developmentPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAYamane, T., Hosen, N., Yamazaki, H., Weissman, I. L.2009; 106 (22): 8953-8958

Abstract

The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.

Abstract

Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.

Abstract

The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

Abstract

Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.

Abstract

We performed the first genome-wide expression analysis directly comparing the expression profile of highly enriched normal human hematopoietic stem cells (HSC) and leukemic stem cells (LSC) from patients with acute myeloid leukemia (AML). Comparing the expression signature of normal HSC to that of LSC, we identified 3,005 differentially expressed genes. Using 2 independent analyses, we identified multiple pathways that are aberrantly regulated in leukemic stem cells compared with normal HSC. Several pathways, including Wnt signaling, MAP Kinase signaling, and Adherens Junction, are well known for their role in cancer development and stem cell biology. Other pathways have not been previously implicated in the regulation of cancer stem cell functions, including Ribosome and T Cell Receptor Signaling pathway. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources applied to highly enriched normal and malignant stem cell populations, can yield an understanding of the critical pathways regulating cancer stem cells.

Abstract

Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fluorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fluorescent protein as a selection marker. The specificity of our custom-designed 7-repeat STAT3 reporter construct was first confirmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confirmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the first study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modified line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.

E2A proteins maintain the hematopoietic stem cell pool and promote the maturation of myelolymphoid and myeloerythroid progenitorsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASemerad, C. L., Mercer, E. M., Inlay, M. A., Weissman, I. L., Murre, C.2009; 106 (6): 1930-1935

Abstract

Hematopoiesis is a tightly controlled process maintained by a small pool of hematopoietic stem cells (HSCs). Here, we demonstrate that the LT-HSC, MPP, premegakaryocytic/erythroid, Pre CFU-E, Pre GM, MkP, and granulocyte-macrophage compartments were all significantly reduced in E2A-deficient bone marrow. Despite a severe depletion of erythroid progenitors, the erythrocyte and megakaryocyte compartments were equivalent in E2A-deficient bone marrow as compared with wild-type mice. E2A-deficient HSCs also failed to efficiently maintain the HSC pool on serial transplantation, and we demonstrate that the E2A proteins regulate cell cycle progression of HSCs by regulating the expression of p21(Cip1), p27(Kip1), and the thrombopoietin receptor, known regulators of HSC self-renewal activity. Based on these observations, we propose that the E2A proteins promote the developmental progression of the entire spectrum of early hematopoietic progenitors and to suppress an erythroid specific program of gene expression in alternative cell lineages. Last, the data mechanistically link E2A, cell cycle regulators, and the maintenance of the HSC pool in a common pathway.

Abstract

Cancer stem cells (CSCs) are defined by their ability to (i) fully recapitulate the tumor of origin when transplanted into immunodeficient mouse hosts, and (ii) self-renew, demonstrated by their ability to be serially transplanted. These properties suggest that CSCs are required for tumor maintenance and metastasis; thus, it has been predicted that CSC elimination is required for cure. This prediction has profoundly altered paradigms for cancer research, compelling investigators to prospectively isolate CSCs to characterize the molecular pathways regulating their behavior. Many potential strategies for CSC-directed therapy have been proposed, but few studies have rigorously demonstrated their efficacy using in vivo models. Herein, we highlight recent studies that demonstrate the utility of CSC-directed therapies and discuss the implications of the CSC hypothesis to experimental design and therapeutic strategies.

Abstract

Ongoing thymopoiesis requires continual seeding from progenitors that reside within the bone marrow (BM), but the identity of the most proximate prethymocytes has remained controversial. Here we take a comprehensive approach to prospectively identify the major source of thymocyte progenitors that reside within the BM and blood, and find that all thymocyte progenitor activity resides within a rare Flk2(+)CD27(+) population. The BM Flk2(+)CD27(+) subset is predominantly composed of common lymphoid progenitors (CLPs) and multipotent progenitors. Of these 2 populations, only CLPs reconstitute thymopoiesis rapidly after intravenous injection. In contrast, multipotent progenitor-derived cells reconstitute the thymus with delayed kinetics only after they have reseeded the BM, self-renewed, and generated CLPs. These results identify CLPs as the major source of thymocyte progenitors within the BM.

Abstract

CX(3)CR1 is a chemokine receptor with a single ligand, the membrane-tethered chemokine CX(3)CL1 (fractalkine). All blood monocytes express CX(3)CR1, but its levels differ between the main 2 subsets, with human CD16(+) and murine Gr1(low) monocytes being CX(3)CR1(hi). Here, we report that absence of either CX(3)CR1 or CX(3)CL1 results in a significant reduction of Gr1(low) blood monocyte levels under both steady-state and inflammatory conditions. Introduction of a Bcl2 transgene restored the wild-type phenotype, suggesting that the CX(3)C axis provides an essential survival signal. Supporting this notion, we show that CX(3)CL1 specifically rescues cultured human monocytes from induced cell death. Human CX(3)CR1 gene polymorphisms are risk factors for atherosclerosis and mice deficient for the CX(3)C receptor or ligand are relatively protected from atherosclerosis development. However, the mechanistic role of CX(3)CR1 in atherogenesis remains unclear. Here, we show that enforced survival of monocytes and plaque-resident phagocytes, including foam cells, restored atherogenesis in CX(3)CR1-deficent mice. The fact that CX(3)CL1-CX(3)CR1 interactions confer an essential survival signal, whose absence leads to increased death of monocytes and/or foam cells, might provide a mechanistic explanation for the role of the CX(3)C chemokine family in atherogenesis.

Abstract

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

Two-step oligoclonal development of male germ cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAUeno, H., Turnbull, B. B., Weissman, I. L.2009; 106 (1): 175-180

Abstract

During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the "deciduous" germ cells are lost, most likely by apoptosis, before the reproductive period. The second "actual" founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.

Abstract

Hematopoietic stem cells (HSCs) have been highly enriched using combinations of 12-14 surface markers. Genes specifically expressed by HSCs as compared with other multipotent progenitors may yield new stem cell enrichment markers, as well as elucidate self-renewal and differentiation mechanisms. We previously reported that multiple cell surface molecules are enriched on mouse HSCs compared with more differentiated progeny. Here, we present a definitive expression profile of the cell adhesion molecule endothelial cell-selective adhesion molecule (Esam1) in hematopoietic cells using reverse transcription-quantitative polymerase chain reaction and flow cytometry studies. We found Esam1 to be highly and selectively expressed by HSCs from mouse bone marrow (BM). Esam1 was also a viable positive HSC marker in fetal, young, and aged mice, as well as in mice of several different strains. In addition, we found robust levels of Esam1 transcripts in purified human HSCs. Esam1(-/-) mice do not exhibit severe hematopoietic defects; however, Esam1(-/-) BM has a greater frequency of HSCs and fewer T cells. HSCs from Esam1(-/-) mice give rise to more granulocyte/monocytes in culture and a higher T cell:B cell ratio upon transplantation into congenic mice. These studies identify Esam1 as a novel, widely applicable HSC-selective marker and suggest that Esam1 may play roles in both HSC proliferation and lineage decisions.

Abstract

Cells of the vertebrate immune system are continuously regenerated by division of hematopoietic stem cells (HSCs) into differentiated effector cells. Classically, HSCs were thought to reside primarily in the bone marrow niche where they produced mature progeny that migrated from the marrow to repopulate the peripheral immune system. However, emerging evidence has established that hematopoietic stem and progenitor cells (HSPCs) are themselves mobile and able to repopulate ectopic niches and contribute more directly to inflammatory responses in the periphery. How the HSPCs remain immune to destruction in a toxic inflammatory milieu is unknown.

Abstract

An effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1(+/-)) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1(+/-) HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1(+/-) HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1(+/-) HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1(+/-) HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell-based therapies.

Abstract

MRI is emerging as a diagnostic modality to track iron-oxide-labeled stem cells. This study investigates whether an off-resonance (OR) pulse sequence designed to generate positive contrast at 1.5T can assess the location, quantity, and viability of delivered stem cells in vivo. Using mouse embryonic stem cell transfected with luciferase reporter gene (luc-mESC), multimodality validation of OR signal was conducted to determine whether engraftment parameters of superparamagnetic iron-oxide labeled luc-mESC (SPIO-luc-mESC) could be determined after cell transplantation into the mouse hindlimb. A significant increase in signal- and contrast-to-noise of the SPIO-luc-mESC was achieved with the OR technique when compared to a gradient recalled echo (GRE) sequence. A significant correlation between the quantity of SPIO-luc-mESC and OR signal was observed immediately after transplantation (R(2) = 0.74, P < 0.05). The assessment of transplanted cell viability by bioluminescence imaging (BLI) showed a significant increase of luciferase activities by day 16, while the MRI signal showed no difference. No significant correlation between BLI and MRI signals of cell viability was observed. In conclusion, using an OR sequence the precise localization and quantitation of SPIO-labeled stem cells in both space and time were possible. However, the OR sequence did not allow evaluation of cell viability.

Abstract

In this work we have uncovered a role for Wnt signaling as an important regulator of stem cell self-renewal in the developing brain. We identified Wnt-responsive cells in the subventricular zone of the developing E14.5 mouse brain. Responding cell populations were enriched for self-renewing stem cells in primary culture, suggesting that Wnt signaling is a hallmark of self-renewing activity in vivo. We also tested whether Wnt signals directly influence neural stem cells. Using inhibitors of the Wnt pathway, we found that Wnt signaling is required for the efficient cloning and expansion of single-cell derived populations that are able to generate new stem cells as well as neurons, astrocytes, and oligodendrocytes. The addition of exogenous Wnt3a protein enhances clonal outgrowth, demonstrating not only a critical role for the Wnt pathway for the regulation of neurogenesis but also its use for the expansion of neural stem cells in cell culture and in tissue engineering.

Abstract

Advances in the understanding of the cells of the hematopoietic system have provided a rich basis for improving clinical hematopoietic cell transplants; finding and using proteins and molecules to amplify or suppress particular blood cell types; understanding the stepwise progression of preleukemic stages leading first to chronic myeloid disorders, then the emergence of acute blastic leukemias; and treating malignant and nonmalignant diseases with cell subsets. As a result of intense scientific investigation, hematopoietic stem cells (HSCs) have been isolated and their key functional characteristics revealed-self-renewal and multilineage differentiation. These characteristics are now found to be present in all tissue/organ stem cell studies, and even in the analysis of pluripotent embryonic, nuclear transfer, and induced pluripotent stem cells. Studies on HSC have identified hematopoiesis as one of the best systems for studying developmental cell lineages and as the best for understanding molecular changes in cell fate decision-making and for finding preclinical and clinical platforms for tissue and organ replacement, regeneration, and oncogenesis. Here we review the steps, from our viewpoint, that led to HSC isolation and its importance in self-nonself immune recognition.

Abstract

Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

Abstract

Individual members of the retinoblastoma (Rb) tumor suppressor gene family serve critical roles in the control of cellular proliferation and differentiation, but the extent of their contributions is masked by redundant and compensatory mechanisms. Here we employed a conditional knockout strategy to simultaneously inactivate all three members, Rb, p107, and p130, in adult hematopoietic stem cells (HSCs). Rb family triple knockout (TKO) mice develop a cell-intrinsic myeloproliferation that originates from hyperproliferative early hematopoietic progenitors and is accompanied by increased apoptosis in lymphoid progenitor populations. Loss of quiescence in the TKO HSC pool is associated with an expansion of these mutant stem cells but also with an enhanced mobilization and an impaired reconstitution potential upon transplantation. The presence of a single p107 allele is sufficient to largely rescue these defects. Thus, Rb family members collectively maintain HSC quiescence and the balance between lymphoid and myeloid cell fates in the hematopoietic system.

Abstract

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.

Abstract

The etiology of the age-associated pathophysiological changes of the hematopoietic system including the onset of anemia, diminished adaptive immune competence, and myelogenous disease development are underwritten by the loss of normal homeostatic control. As tissue and organ homeostasis in adults is primarily mediated by the activity of stem and progenitor cells, it has been suggested that the imbalances accompanying aging of the hematopoietic system may stem from alterations in the prevalence and/or functional capacity of hematopoietic stem cells (HSCs) and progenitors. In this review, we examine evidence implicating a role for stem cells in the aging of the hematopoietic system, and focus on the mechanisms suggested to contribute to stem cell aging.

Abstract

Dr. Irving Weissman was the honored E. Donnall Thomas lecturer at the Tandem BMT Meetings, held on February 10, 2007, at Keystone, Colorado. Dr. Weissman has been a major player, and has provided us with enormous insight into many areas of biology, dating back to his high school days in Montana. He led an enormously productive career at Stanford University where he has taught us many lessons involving our understanding of lymphocyte homing, stem cell biology, both of the hematopoietic system and other types of stem cells, and also now, about cancer stem cells. Dr. Weissman has made enormous contributions to this burgeoning field that has provided us new insights and new opportunities for treatment strategies. In addition to a very productive laboratory career, he is also currently the director of both the Stem Cell Institute, as well as the Cancer Center at Stanford University. The following text is a modified transcribed version of the presentation made by Dr. Weissman.

Abstract

The mammalian blood system contains a multitude of distinct mature cell lineages adapted to serving diverse functional roles. Mutations that abrogate the development or function of one or more of these lineages can lead to profound adverse consequences, such as immunodeficiency, autoimmunity, or anemia. Replacement of hematopoietic stem cells (HSC) that carry such mutations with HSC from a healthy donor can reverse such disorders, but because the risks associated with the procedure are often more serious than the blood disorders themselves, bone marrow transplantation is generally not used to treat a number of relatively common inherited blood diseases. Aside from a number of other problems, risks associated with cytoreductive treatments that create "space" for donor HSC, and the slow kinetics with which immune competence is restored following transplantation hamper progress. This review will focus on how recent studies using experimental model systems may direct future efforts to implement routine use of HSC transplantation to cure inherited blood disorders.

Abstract

We tested the hypothesis that multimodality imaging of mouse embryonic stem cells (mESCs) provides accurate assessment of cellular location, viability, and restorative potential after transplantation into different zones of myocardial infarction.Mice underwent left anterior descending artery ligation followed by transplantation of dual-labeled mESCs with superparamagnetic iron oxide and luciferase via direct injection into 3 different zones of myocardial infarction: intra-infarction, peri-infarction, and normal (remote). One day after transplantation, magnetic resonance imaging enabled assessment of the precise anatomic locations of mESCs. Bioluminescence imaging allowed longitudinal analysis of cell viability through detection of luciferase activity. Subsequent evaluation of myocardial regeneration and functional restoration was performed by echocardiography and pressure-volume loop analysis. Using 16-segment analysis, we demonstrated precise localization of dual-labeled mESCs. A strong correlation between histology and magnetic resonance imaging was established (r=0.962, P=0.002). Bioluminescent imaging data demonstrated that cell viability in the remote group was significantly higher than in other groups. Echocardiography and pressure-volume loop analysis revealed improved functional restoration in animals treated with mESCs, although myocardial regeneration was not observed.Multimodality evaluation of mESC engraftment in the heterogeneous tissue of myocardial infarction is possible. Magnetic resonance imaging demonstrated accurate anatomic localization of dual-labeled mESCs. Bioluminescent imaging enabled assessment of variable viability of mESCs transplanted into the infarcted myocardium. Echocardiography and pressure-volume loop analysis validated the restorative potential of mESCs. Although mESCs transplanted into the remote zone demonstrated the highest viability, precise delivery of mESCs into the peri-infarction region might be equally critical in restoring the injured myocardium.

Abstract

Mature blood cells develop from multipotent hematopoietic stem cells through a series of sequential intermediates in which the developmental potential for particular blood lineages is progressively extinguished. We previously reported the identification of one of these developmental intermediates, the common lymphoid progenitor (CLP), which can give rise to T cells, B cells, dendritic cells (DCs), and natural killer cells (NKs), but lacks myeloid and erythroid potential. Recently, several studies have suggested that the T-cell and DC potential of CLP is limited or absent, and/or that CLP contains significant myeloid potential. Here, we show that the originally identified CLP population can be divided into functionally distinct subsets based on the expression of the tyrosine kinase receptor, Flk2. The Flk2(+) subset contains robust in vivo and in vitro T-cell, B-cell, DC, and NK potential, but lacks myeloid potential and, therefore, represents an oligopotent, lymphoid-restricted progenitor. This population of cells does not appear to be B cell-biased and robustly reconstitutes both B and T lineages in vivo, consistent with its being a physiologic progenitor of both of these subsets. Thus, Flk2 expression defines a homogeneous, readily obtainable subset of bone marrow CLP that is completely lymphoid-committed and can differentiate equivalently well into both B and T lineages.

Abstract

Liver progenitor cells, characterized by the coexpression of biliary and hepatocyte lineage markers and the ability to form colonies in culture, were isolated by flow cytometry from primary human fetal livers. These prospectively isolated liver progenitor cells supported hepatitis D virus infection, expressed, and produced albumin and alpha-fetoprotein, as tracked by albumin- and alpha-fetoprotein-driven lentiviral promoter reporter constructs and measured by ELISA, respectively. Coculture in three-dimensional (3D) fibrin gel with endothelial cells resulted in the formation of vascular structures by the endothelial cells and increased proliferation of liver progenitors. The enhanced proliferation of liver progenitors that was observed when liver progenitors and endothelial cells were cultured in direct contact was not achieved when liver progenitors and endothelial cells were cultured on adjacent but separate matrices and when they were cultured across transwell membranes. In conclusion, coculture of liver progenitors and endothelial cells in three-dimensional matrix resulted in enhanced liver progenitor proliferation and function. This coculture methodology offers a novel coculture system that could be applied for the development of engineered liver tissues.

Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growthPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAPurhonen, S., Palm, J., Rossi, D., Kaskenpaa, N., Rajantie, I., Yla-Herttuala, S., Alitalo, K., Weissman, I. L., Salven, P.2008; 105 (18): 6620-6625

Abstract

The mechanisms by which bone marrow (BM)-derived stem cells might contribute to angiogenesis and the origin of neovascular endothelial cells (ECs) are controversial. Neovascular ECs have been proposed to originate from VEGF receptor 2-expressing (VEGFR-2+) stem cells mobilized from the BM by VEGF or tumors, and it is thought that angiogenesis and tumor growth may depend on such endothelial precursors or progenitors. We studied the mobilization of BM cells to circulation by inoculating mice with VEGF polypeptides, adenoviral vectors expressing VEGF, or tumors. We induced angiogenesis by syngeneic melanomas, APCmin adenomas, adenoviral VEGF delivery, or matrigel plugs in four different genetically tagged universal or endothelial cell-specific chimeric mouse models, and subsequently analyzed the contribution of BM-derived cells to endothelium in a wide range of time points. To study the existence of circulating ECs in a nonmyeloablative setting, pairs of genetically marked parabiotic mice with a shared anastomosed circulatory system were created. We did not observe specific mobilization of VEGFR-2+ cells to circulation by VEGF or tumors. During angiogenesis, abundant BM-derived perivascular cells were recruited close to blood vessel wall ECs but did not form part of the endothelium. No circulation-derived vascular ECs were observed in the parabiosis experiments. Our results show that no BM-derived VEGFR-2+ or other EC precursors contribute to vascular endothelium and that cancer growth does not require BM-derived endothelial progenitors. Endothelial differentiation is not a typical in vivo function of normal BM-derived stem cells in adults, and it has to be an extremely rare event if it occurs at all.

Abstract

SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice also showed increased growth capacity and decreased dependency on growth factors. Consistent with this, SIRT1 inhibition was associated with increased telomerase activity in human cells. We also observed a significant increase in AMPK levels up on SIRT1 inhibition under glucose limiting conditions. Although SIRT1 suppression cooperated with hTERT to promote cell growth, either overexpression or suppression of SIRT1 alone had no effect on life span of human diploid fibroblasts. Our findings challenge certain models and connect nutrient sensing enzymes to the immortalization process. Furthermore, they show that in certain cell lineages, SIRT1 can act as a growth suppressor gene.

Abstract

The aging of tissue-specific stem cell and progenitor cell compartments is believed to be central to the decline of tissue and organ integrity and function in the elderly. Here, we examine evidence linking stem cell dysfunction to the pathophysiological conditions accompanying aging, focusing on the mechanisms underlying stem cell decline and their contribution to disease pathogenesis.

Abstract

Bone marrow-derived cells are recruited into tumor vasculature in response to angiogenic signals, and some of the cells within the newly forming tumor vessels are hematopoietic stem cells (HSCs) in origin. Previous studies suggest that bone marrow-derived pericytes are associated with newly formed vessels in tumors. In this study, we used an orthotopic rat glioma model (RT-2/RAG) to examine the contribution of long-term hematopoietic stem cell (LT-HSC)-derived pericytic cells to brain tumor angiogenesis. Mice (RAG-2/KO5.2) were lethally irradiated, and their hematopoietic cells were repopulated by transplantation of double fluorescence-activated cell-sorted LT-HSCs that express green fluorescent protein (GFP+). RT-2/RAG cells were then injected into the striatum of the chimeric mice 6 weeks post-transplantation. The animals were sacrificed 9 days after tumor implantation, and the incorporation and lineage-specific marker expression profile of the GFP+ cells within the growing tumor and tumor periphery were analyzed. LT-HSC-derived GFP+ cells were noted to incorporate onto the surface of tumor vessels within the perivascular space. LT-HSC-derived GFP+ cells express the pericyte progenitor marker, platelet-derived growth factor receptor-beta (PDGFR beta), as well as mature perictyte markers such as nerve/glial antigen 2 proteoglycan (NG2), alpha-smooth muscle actin (alpha SMA), and desmin. These LT-HSC-derived cells may represent a population of progenitor or committed pericytes within the neovascular tree and may play a role in shaping the angio-architecture in the vascular niche of brain tumors.

Abstract

Many types of adult tissues, especially for high turnover tissues such as the blood and intestinal system, stand on a hierarchical tissue-specific stem cell system. Tissue-specific stem cells concurrently have self-renewal capacity and potential to give rise to all types of mature cells in their tissue. The differentiation process of the tissue-specific stem cell is successive restriction of these capacities. The first progeny of tissue-specific stem cells are multipotent progenitors (MPPs) that lose long-term self-renewal capacity yet have full lineage potential. MPPs in turn give rise to oligopotent progenitors, which then commit into lineage-restricted progenitors. This hierarchical system enables a lifelong supply of matured functional cells that generally have a short life span and a relatively high turnover rate. In this chapter, we review our findings and other key experiments that have led to the establishment of the current cellular stem and progenitor hierarchy in the blood-forming systems of mice and humans for both normal and leukemic hematopoiesis. We also review select signaling pathways intrinsic to normal hematopoietic and leukemic stem cell populations as well our recent findings elucidating the possible origin of the leukemia stem cell.

Abstract

Establishment of robust xenograft models is critical to studying human hematopoiesis in a physiologic setting. Using a recently developed immunodeficient mouse strain, we have established long-term multilineage human grafts and demonstrated their serially transplantability using limited numbers of purified human hematopoietic stem cells (HSCs). Herein, we describe our protocol for the isolation of human HSC (Lin-CD34+CD38-CD90+) from umbilical cord blood (CB) as well as the xenotransplantation system that allows stable engraftment of human hematopoietic cells with as few as ten HSCs. Isolation of CB mononuclear cells requires 2-3 h, and cells may be cryopreserved before transplantation. Isolation of HSC requires approximately 2-3 h, and transplantation requires 1 h. Short-term and long-term engraftment is assessed 4-6 weeks and 10-12 weeks post-transplantation, respectively, with preparation and analysis time requiring 4-8 h at each time point.

Abstract

Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.

Abstract

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.

Abstract

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

Abstract

It has been proposed that cumulative somatic mutations contribute to the aging process by disrupting the transcriptional networks that regulate cell structure and function. Experimental support for this model emerged from a recent study of cardiomyocytes that showed a dramatic increase in the transcriptional heterogeneity of these long-lived postmitotic cells with age. To determine if regulatory instability is a hallmark of aging in renewing tissues, we evaluated gene expression noise in four hematopoietic cell types: stem cells, granulocytes, naïve B cells and naïve T cells. We used flow cytometry to purify phenotypically equivalent cells from young and old mice, and applied multiplexed quantitative reverse transcription-polymerase chain reaction to measure the copy number of six different mRNA transcripts in 324 individual cells. There was a trend toward higher transcript levels in cells isolated from old animals, but no significant increase in transcriptional heterogeneity with age was found in the surveyed populations. Flow cytometric analysis of membrane protein expression also indicated that cell-to-cell variability was unaffected by age. We conclude that large-scale regulatory destabilization is not a universal concomitant of aging, and may be of significance as an aging mechanism primarily in nonrenewing tissues.

Abstract

Upon intravenous transplantation, hematopoietic stem cells (HSCs) can home to specialized niches, yet most HSCs fail to engraft unless recipients are subjected to toxic preconditioning. We provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Administration of ACK2, an antibody that blocks c-kit function, led to the transient removal of >98% of endogenous HSCs in immunodeficient mice. Subsequent transplantation of these mice with donor HSCs led to chimerism levels of up to 90%. Extrapolation of these methods to humans may enable mild but effective conditioning regimens for transplantation.

Abstract

Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure.

Abstract

The aging of tissue-specific stem and progenitor cells is believed to be central to the pathophysiological conditions arising in aged individuals. While the mechanisms driving stem cell aging are poorly understood, mounting evidence points to age-dependent DNA damage accrual as an important contributing factor. While it has been postulated that DNA damage may deplete stem cell numbers with age, recent studies indicate that murine hematopoietic stem cell (HSC) reserves are in fact maintained despite the accrual of genomic damage with age. Evidence suggests this to be a result of the quiescent (G0) cell cycle status of HSC, which results in an attenuation of checkpoint control and DNA damage responses for repair or apoptosis. When aged stem cells that have acquired damage are called into cycle under conditions of stress or tissue regeneration however, their functional capacity was shown to be severely impaired. These data suggest that age-dependent DNA damage accumulation may underlie the diminished capacity of aged stem cells to mediate a return to homeostasis after acute stress or injury. Moreover, the cytoprotection afforded by stem cell quiescence in stress-free, steady-state conditions suggests a mechanism through which potentially dangerous lesions can accumulate in the stem cell pool with age.

Abstract

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

Abstract

Cancer stem cells (CSCs) are cells that drive tumorigenesis, as well as giving rise to a large population of differentiated progeny that make up the bulk of the tumor, but that lack tumorigenic potential. CSCs have been identified in a variety of human tumors, as assayed by their ability to initiate tumor growth in immunocompromised mice. Further characterization studies have demonstrated that gene expression profiles in breast cancer correlate with patient prognosis, and brain CSCs are specifically resistant to radiation through DNA damage repair. In addition, specific signaling pathways play a functional role in CSC self renewal and/or differentiation, and early studies indicate that CSCs are associated with a microenvironmental niche. Thus the biological properties of CSCs are just beginning to be revealed, and the continuation of these studies should lead to the development of CSC-targeted therapies for cancer treatment.

Abstract

Hematopoietic stem cells (HSCs) divide and give rise to more committed progenitors, which ultimately produce all lineages of blood cells. HSCs can be induced to enter the cell cycle in vitro and in vivo by stimulatory cytokines and in vivo by ablation of bone marrow (BM) cells with irradiation or chemotherapeutic agents. Although it has been postulated that rates of HSC proliferation increase with normal hematopoietic stresses, such as infection or hemorrhage, this hypothesis has never been directly tested. The ability to analyze HSCs prospectively by cell-surface phenotype c-kit(+), Thy1.1(lo), Sca-1(+), Linage(neg/lo) has allowed us to perform a detailed examination of the effects of bleeding on the cell cycle kinetics of HSCs. Our results demonstrate for the first time that HSCs in both the BM and the spleen proliferate and self-renew in response to tail-vein bleeding in mice. This response was suppressed when red blood cells, but not when white blood cells, were transferred after bleeding. Thus, regulators of HSC proliferation can sense and respond to red blood cell levels.

Abstract

Patients with severe systemic lupus erythematosus (SLE) refractory to conventional treatment are candidates for autologous hematopoietic stem cell (HSC) transplantation if the intent is to reset the immunologic clock. These patients might be candidates for allotransplantation with (SLE)-resistant major histocompatibility complex (MHC) haplotype-matched HSC if partial or complete replacement of an autoimmune-prone system is the intent. Using lupus-prone New Zealand black x New Zealand white (NZBW) mice, we investigated the use of highly enriched, haplomismatched, allogeneic HSC to prevent development of or to treat established autoimmune pathology. Young NZBW mice receiving purified allogeneic HSC transplants had improved survival, decreased proteinuria, circulating immune complexes, and autoantibodies to nuclear antigens than did untreated mice or mice given NZBW HSCs. NZBW mice with established lupus-like disease that received nonmyeloablative conditioning and transplants of (MHC) haplomismatched allogeneic HSCs also had greatly increased overall survival. Mice that received transplants exhibited stabilization or reversal of their lupus symptoms; stabilized or decreased proteinuria, and a lower frequency of elevated circulating immune complexes or autoantibodies than did control mice. Induction of durable mixed chimerism by transplantation of purified allogeneic HSCs after nonmyeloablative conditioning has the potential to reverse symptoms of established NZBW lupus.

Abstract

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

Abstract

Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.

Abstract

Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state.

Abstract

The factors that regulate the rate of production of T cells by the thymus remain incompletely defined. To test whether generation of functional T cell receptors limits the rate of thymic T cell export, we made use of a line of mice, LN3alphabeta, that have endogenously prerearranged TCR genes. The prerearranged TCR genes were expressed abnormally early in hemopoietic development, indicating that RAG-mediated recombination, rather than transcription factor expression, is the key determinant of the initiation of robust TCR transcription. Thymic T cell export rates were similar between wild-type (wt) and LN3alphabeta mice, indicating that T cell maturation rates in these mice are determined by factors other than TCR gene rearrangement. In competitive bone marrow chimeras, however, LN3alphabeta thymocytes were out-competed by wt cells and failed to develop beyond the double-negative 4 stage. Furthermore, wt progenitors transplanted intrathymically into LN3alphabeta mice proliferated excessively, suggesting that increased proliferative signals in the LN3alphabeta thymus compensate for faulty T cell development driven by early TCR expression.

Abstract

The ability to self-renew is essential for all kinds of stem cells regardless of tissue type. One of the best candidate genes involved in conferring self-renewal capacity is Bmi-1, which has been proven to be essential for the maintenance of both normal adult hematopoietic and leukemia stem cells, as well as adult neural stem cells. To investigate the possible role of Bmi-1 in other cell types that also self-renew, we generated Bmi-1-green fluorescent protein (GFP)-knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of the Bmi-1 gene. Using these targeted reporter mice, we demonstrated that Bmi-1 is expressed in hematopoietic stem cells (HSCs) at its highest levels and downregulated upon commitment to differentiation. An in vivo reconstitution assay revealed that the frequency of HSCs was 1/16 in Bmi-1high c-kit+ lin -Sca-1+ bone marrow (BM) cells and 1/49 in Bmi-1 high lin- BM cells, suggesting that Bmi-1 may serve as a marker for normal HSCs. In murine leukemia models induced by P210BCR/ABL or TEL/PDGFbetaR + AML1/ETO, Bmi-1 was not overexpressed in leukemic HSCs, despite the increase in the HSC numbers. Bmi-1 was expressed at its highest levels in undifferentiated leukemia cells. Furthermore, in several other nonhematopoietic tissues, cells could be separated into distinct subpopulations with differential Bmi-1 expression. Thus, these mice allow for the isolation of viable Bmi-1-expressing cells and have the potential to become a useful tool for understanding the role of Bmi-1 in normal and cancer stem cells in multiple tissue types. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34(+)CD38(-) AML cells in many cases (74.0 +/- 25.3% in 19 of 29 cases), whereas only a few (4.9 +/- 1.6%) cells in the normal HSC-enriched population (Lin(-)CD34(+)CD38(-)CD90(+)) expressed CD96 weakly. To examine whether CD96(+) AML cells are enriched for LSC activity, we separated AML cells into CD96(+) and CD96(-) fractions and transplanted them into irradiated newborn Rag2(-/-) gamma(c)(-/-) mice. In four of five samples, only CD96(+) cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

Abstract

A diminished capacity to maintain tissue homeostasis is a central physiological characteristic of ageing. As stem cells regulate tissue homeostasis, depletion of stem cell reserves and/or diminished stem cell function have been postulated to contribute to ageing. It has further been suggested that accumulated DNA damage could be a principal mechanism underlying age-dependent stem cell decline. We have tested these hypotheses by examining haematopoietic stem cell reserves and function with age in mice deficient in several genomic maintenance pathways including nucleotide excision repair, telomere maintenance and non-homologous end-joining. Here we show that although deficiencies in these pathways did not deplete stem cell reserves with age, stem cell functional capacity was severely affected under conditions of stress, leading to loss of reconstitution and proliferative potential, diminished self-renewal, increased apoptosis and, ultimately, functional exhaustion. Moreover, we provide evidence that endogenous DNA damage accumulates with age in wild-type stem cells. These data are consistent with DNA damage accrual being a physiological mechanism of stem cell ageing that may contribute to the diminished capacity of aged tissues to return to homeostasis after exposure to acute stress or injury.

Abstract

Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B-cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19(+)) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B-cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.

Abstract

Embryogenesis and asexual reproduction are commonly considered to be coordinated developmental processes, which depend on accurate progression through a defined sequence of developmental stages. Here we report a peculiar developmental scenario in a simple chordate, Botryllus schlosseri, wherein a normal colony of individuals (zooids and buds) is regenerated from the vasculature (vascular budding) through a sequence of morphologically abnormal developmental stages. Vascular budding was induced by surgically removing buds and zooids from B. schlosseri colonies, leaving only the vasculature and the tunic that connects them. In vivo imaging and histological sections showed that the timing and morphology of developing structures during vascular budding deviated significantly from other asexual reproduction modes (the regular asexual reproduction mode in this organism and vascular budding in other botryllid species). Subsequent asexual reproduction cycles exhibited gradual regaining of normal developmental patterns, eventually leading to regeneration of a normal colony. The conversion into a normal body form suggests the activation of an alternative pathway of asexual reproduction, which involves gradual regaining of normal positional information. It presents a powerful model for studying the specification of the same body plan by different developmental programs.

Abstract

Advancing age is frequented by the onset of a variety of hematological conditions characterized by diminished homeostatic control of blood cell production. The fact that upstream hematopoietic stem and progenitor cells are obligate mediators of homeostatic control of all blood lineages, has implicated the involvement of these cells in the pathophysiology of these conditions. Indeed, evidence from our group and others has suggested that two of the most clinically significant age-associated hematological conditions, namely, the diminution of the adaptive immune system and the elevated incidence of myeloproliferative diseases, have their origin in cell autonomous changes in the functional capacity of hematopoietic stem cells.

Abstract

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40-80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 10(3)-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Abstract

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.

Abstract

Differentiated cell types derived from human embryonic stem cells (hESCs) may serve in the future to treat various human diseases and to model early human embryonic development in vitro. Fulfilling this potential, however, requires extensive development of methods and reagents for studying hESCs self-renewal and differentiation. One of the most widely used experimental approaches in the field of stem cell research is the identification of cell surface markers that can be used to prospectively define and isolate specific populations of stem cells and their progenitors. Here, we review an efficient method for generating monoclonal antibodies against cell surface antigens expressed by hESCs and stem cells at different stages of differentiation. This method may have profound implications for many aspects of hESC research and therapeutics.

Abstract

There is a growing body of evidence that supports the idea that malignant tumors are initiated and maintained by a population of tumor cells that share similar biologic properties to normal adult stem cells. This model, the cancer stem cell (CSC) hypothesis, is based on the observation that tumors, like adult tissues, arise from cells that exhibit the ability to self-renew as well as give rise to differentiated tissue cells. Although the concept of the CSC is not entirely new, advances made over the past two decades in our understanding of normal stem cell biology in conjunction with the recent application of these concepts to experimentally define CSCs have resulted in the identification of CSCs in several human malignancies.

Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCRPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWarren, L., Bryder, D., Weissman, I. L., Quake, S. R.2006; 103 (47): 17807-17812

Abstract

We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.

Abstract

Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event.

Abstract

Neoangiogenesis is a prerequisite for the full phenotypic expression and growth of a malignant tumor mass. It is believed to be triggered by tissue hypoxia and involves proliferation and sprouting of the preexisting vessels and the recruitment of endothelial progenitor cells from bone marrow.A chimeric mouse model was used to examine the contribution of these progenitor cells to the neovasculature of brain tumor. T-cell knockout (RAG/KO5.2) mice were irradiated lethally, and their bone marrow was repopulated with T-cell depleted green fluorescent protein (GFP)-expressing bone marrow cells. RAG/RT-2 glioma cells were implanted into the striatum of the animals. Neovascular formation at various times of tumor growth was monitored together with the extent of incorporation of GFP+ bone marrow-derived cells within the vascular tree, in particular, cells carrying the endothelial progenitor markers CD34 and Flk-1.The recruitment of GFP+ cells to the growing tumor and their incorporation into the vascular network occurred during the period of increasing vascular density and preceded the expansion of the tumor. The number of marrow-derived cells with endothelial morphology and phenotype was small but significant (4% of all endothelial cells at Day 12); 54% of all tumor vessels contained at least one GFP+ cell.Our results suggest that bone marrow cells are recruited to newly formed and remodeled tumor vessels. Their recruitment may occur in response to signals from a highly proliferating milieu, and their role is to support the neovascular complex and to promote tumor growth.

Abstract

The recent prospective isolation of a wide variety of somatically derived stem cells has affirmed the notion that homeostatic maintenance of most tissues and organs is mediated by tissue-specific stem and progenitor cells and fueled enthusiasm for the use of such cells in strategies aimed at repairing or replacing damaged, diseased, or genetically deficient tissues and organs. Hematopoietic stem cells (HSCs) are arguably the most well-characterized tissue-specific stem cell, with decades of basic research and clinical application providing not only a profound understanding of the principles of stem cell biology, but also of its potential pitfalls. It is our belief that emerging stem cell fields can benefit greatly from an understanding of the lessons learned from the study of HSCs. In this review we discuss some general concepts regarding stem cell biology learned from the study of HSCs with a highlight on recent work pertaining to emerging topics of interest for stem cell biology.

Abstract

A model of hematopoietic development wherein multipotentiality is conserved until segregation of myeloid and lymphoid potential has recently been challenged, proposing that megakaryocyte/erythrocyte (MegE) potential is lost in Flk2/Flt3-expressing early progenitors. Here, we used sensitive in vivo approaches to quantitatively and kinetically assess the MegE potential of hematopoietic stem cells and various Flk2(+) early progenitors and compared it with the MegE potential of downstream committed myeloid and lymphoid progenitors and with their ability to give rise to mature myelomonocytic and lymphoid cells. We demonstrate that Flk2(+) early progenitors retain MegE potential in vivo both at the population and clonal levels. These results indicate that Flk2 expression by early progenitors is not at the expense of full multipotency and support the current model of hematopoietic development with segregation of myeloid and lymphoid lineages from multipotent progenitors.

Abstract

Histocompatibility in the primitive chordate, Botryllus schlosseri, is controlled by a single, highly polymorphic locus, the FuHC. By taking a forward genetic approach, we have identified a locus encoded near the FuHC, called fester, which is polymorphic, polygenic, and inherited in distinct haplotypes. Somatic diversification occurs through extensive alternative splicing, with each individual expressing a unique repertoire of splice forms, both membrane bound and potentially secreted, all expressed in tissues intimately associated with histocompatibility. Functional studies, via both siRNA-mediated knockdown and direct blocking by monoclonal antibodies raised against fester, were able to disrupt predicted histocompatibility outcomes. The genetic and somatic diversity, coupled to the expression and functional data, suggests that fester is a receptor involved in histocompatibility.

Abstract

The replacement of abnormal hematopoietic stem cells (HSCs) with normal transplanted HSCs can correct a wide range of hematologic disorders. Here, we provide evidence that transplantation of more differentiated progenitor cells can be used to more rapidly correct lymphoid deficiencies in unconditioned immunocompromised mice. Transplantation of flk2+ multipotent progenitors led to robust B and T cell reconstitution that was maintained for at least 16 weeks. Antigenic challenge at 16 weeks post-transplantation revealed that reconstituted lymphocytes maintained a functional repertoire. In contrast to the persistent lymphocytic engraftment, myeloid chimerism was lost by 12 weeks post-transplantation consistent with the fact that flk2+ progenitors are non-self-renewing. Thus, while more differentiated progenitors are capable of rescuing lymphoid deficiencies, transplantation of HSCs must be used for the correction of non-lymphoid disorders, and, we propose, very long-term immune reconstitution. Based on recent evidence, we discuss novel strategies to achieve the replacement of abnormal HSCs without the use of cytotoxic conditioning regimens.

Abstract

Self-renewal pathways crucial for maintaining stem cells are deregulated in cancer, raising the spectre that cancer therapies targeting such pathways might also ablate normal stem cells. As Yilmaz et al. (2006) report in a recent Nature paper, this may not be the case for the tumor suppressor protein Pten, which drives the self-renewal of normal hematopoietic stem cells and the formation of leukemia cells through different mechanisms.

Abstract

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALuckey, C. J., Bhattacharya, D., Goldrath, A. W., Weissman, I. L., Benoist, C., MATHIS, D.2006; 103 (9): 3304-3309

Abstract

The only cells of the hematopoietic system that undergo self-renewal for the lifetime of the organism are long-term hematopoietic stem cells and memory T and B cells. To determine whether there is a shared transcriptional program among these self-renewing populations, we first compared the gene-expression profiles of naïve, effector and memory CD8(+) T cells with those of long-term hematopoietic stem cells, short-term hematopoietic stem cells, and lineage-committed progenitors. Transcripts augmented in memory CD8(+) T cells relative to naïve and effector T cells were selectively enriched in long-term hematopoietic stem cells and were progressively lost in their short-term and lineage-committed counterparts. Furthermore, transcripts selectively decreased in memory CD8(+) T cells were selectively down-regulated in long-term hematopoietic stem cells and progressively increased with differentiation. To confirm that this pattern was a general property of immunologic memory, we turned to independently generated gene expression profiles of memory, naïve, germinal center, and plasma B cells. Once again, memory-enriched and -depleted transcripts were also appropriately augmented and diminished in long-term hematopoietic stem cells, and their expression correlated with progressive loss of self-renewal function. Thus, there appears to be a common signature of both up- and down-regulated transcripts shared between memory T cells, memory B cells, and long-term hematopoietic stem cells. This signature was not consistently enriched in neural or embryonic stem cell populations and, therefore, appears to be restricted to the hematopoeitic system. These observations provide evidence that the shared phenotype of self-renewal in the hematopoietic system is linked at the molecular level.

Abstract

Langerhans cells (LCs) are antigen-presenting cells (APCs) residing in the epidermis that play a major role in skin immunity. Our earlier studies showed that when skin is inflamed LCs are replaced by bone marrow-derived progenitor cells, while during steady-state conditions LCs are able to self-renew in the skin. Identification of the LC progenitors in bone marrow would represent a critical step toward identifying the factors that regulate LC generation as well as their trafficking to the skin. To determine LC lineage origin, we reconstituted lethally irradiated CD45.2 mice with rigorously purified lymphoid and myeloid progenitors from CD45.1 congenic mice. Twenty-four hours later, we exposed the mice to UV light to deplete resident LCs and induce their replacement by progenitors. Reconstitution with common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs), or early thymic progenitors led to LC generation within 2 to 3 weeks. CMPs were at least 20 times more efficient at generating LCs than CLPs. LCs from both lineages were derived almost entirely from fetal liver kinase-2+ (Flk-2+) progenitors, displayed typical dendritic-cell (DC) morphology, and showed long-term persistence in the skin. These results indicate that LCs are derived mainly from myeloid progenitors and are dependent on Flt3-ligand for their development.

Abstract

Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegc-kitposSca-1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c-kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%-70% of the cells in BFU-E-derived colonies but are not present among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10- to 20-fold under acute and chronic stress, whereas MEP cell increases (21%-84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream-downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented.

Abstract

In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoietic stem cells (HSCs) are thought to fill the unique niches within the bone marrow that allow maintenance of full hematopoietic potential and thus prevent productive engraftment of transplanted donor HSCs. By transplantation of purified exogenous HSCs into unconditioned congenic histocompatible strains of mice, we show that approximately 0.1-1.0% of these HSC niches are available for engraftment at any given point and find no evidence that endogenous HSCs can be displaced from the niches they occupy. We demonstrate that productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells that recognize very subtle minor histocompatibility differences. Strikingly, transplantation of purified HSCs into a panel of severe combined immunodeficient (SCID) mice leads to a rapid and complete rescue of lymphoid deficiencies through engraftment of these very rare niches and expansion of donor lymphoid progenitors. We further demonstrate that transient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a general mechanism by which transplanted HSCs can correct hematopoietic deficiencies without any host conditioning or with only highly specific and transient lymphoablation.

Abstract

This review focuses on the genomics of mouse hematopoiesis, but also draws parallels to other systems and discusses issues common to the analysis of rare populations such as stem cells. As examples from the mouse blood forming system are used to illustrate several points, the authors first give a brief introduction to mouse hematopoiesis as a model system. We review the multiple microarray analyses that have been performed on various mouse hematopoietic subpopulations and comment on both technical and biological aspects of such experiments. The concept of stemness is discussed, and the importance of biological function of gene products, protein-protein interactions and molecular pathways highlighted. Finally, the authors discuss some major unresolved issues in hematopoiesis and discuss the potential uses of future microarray analysis as well as other genomic and functional approaches that might prove useful to further our understanding of hematopoiesis and other stem cell systems.

Abstract

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.

Abstract

Knowledge of the molecular networks controlling the proliferation and fate of hematopoietic stem cells (HSC) is essential to understand their function in maintaining blood cell production during normal hematopoiesis and upon clinical transplantation. Using highly purified stem and progenitor cell populations, we define the proliferation index and status of the cell cycle machinery at discrete stages of hematopoietic differentiation and during cytokine-mediated HSC mobilization. We identify distinct sets of cell cycle proteins that specifically associate with differentiation, self-renewal, and maintenance of quiescence in HSC and progenitor cells. Moreover, we describe a striking inequality of function among in vivo cycling and quiescent HSC by demonstrating that their long-term engraftment potential resides predominantly in the G(0) fraction. These data provide a direct link between HSC proliferation and function and identify discrete molecular targets in regulating HSC cell fate decisions that could have implications for both the therapeutic use of HSC and the understanding of leukemic transformation.

Abstract

Development via regeneration or budding shares some known genetic pathways with embryogenesis, but no concerted effort has been made to identify genes unique to asexual development. We have identified a novel gene that plays a role in cyclical bud formation and asexual organogenesis in the colonial ascidian Botryllus schlosseri. Athena mRNA is transcribed at high levels during the 24- to 36-hr interval of programmed cell death and new bud initiation at the conclusion of the budding cycle (takeover). Knockdown of Athena by RNAi and antisense morpholinos induced defects in the development of new buds ranging from retardation in growth and abnormal organogenesis to hollow buds lacking organs. As genetic intervention in this organism has not been possible, this study establishes the use of RNAi and morpholinos in Botryllus as well as describing the knockdown phenotype of a new gene.

Abstract

Histocompatibility--the ability of an organism to distinguish its own cells and tissue from those of another--is a universal phenomenon in the Metazoa. In vertebrates, histocompatibility is a function of the immune system controlled by a highly polymorphic major histocompatibility complex (MHC), which encodes proteins that target foreign molecules for immune cell recognition. The association of the MHC and immune function suggests an evolutionary relationship between metazoan histocompatibility and the origins of vertebrate immunity. However, the MHC of vertebrates is the only functionally characterized histocompatibility system; the mechanisms underlying this process in non-vertebrates are unknown. A primitive chordate, the ascidian Botryllus schlosseri, also undergoes a histocompatibility reaction controlled by a highly polymorphic locus. Here we describe the isolation of a candidate gene encoding an immunoglobulin superfamily member that, by itself, predicts the outcome of histocompatibility reactions. This is the first non-vertebrate histocompatibility gene described, and may provide insights into the evolution of vertebrate adaptive immunity.

Abstract

The goal of this study was to illustrate the findings of a significant subpopulation of cells within a pediatric astroblastoma that have the specific cell surface phenotype found on known human neural stem cells.Cells with a cell surface marker profile characteristic of human neural stem cells were isolated using fluorescence-activated cell sorting from a mostly nonmitotic astroblastoma removed from the brain of an 11-year-old girl. An unusually high proportion (24%) of the cells were CD133 positive and CD24, CD34, and CD45 negative (CD133(+)CD24(-)CD34(-)CD45(-) cells), the phenotypic antigenic pattern associated with neural stem cells; very few CD133-positive cells were not also CD24, CD34, and CD45 negative. Some cells (12%) were CD34 positive, indicating the presence within the tumor of hematopoietic stem cells. Cells formed cytospheres that resembled neurospheres when seeded into stem cell media and coexpressed beta-tubulin and glial fibrillary acidic protein (GFAP) but did not express the oligodendrocyte marker O4. Cell proliferation was demonstrated by incorporation of bromodeoxyuridine. The cells lost their capacity for self-renewal in vitro after four to six passages, although they continued to coexpress beta-tubulin and GFAP. The cells did not differentiate into neurons or astrocytes when placed in differentiation medium.Although this astroblastoma contained a high proportion of phenotypic neural stemlike cells, the cells had limited proliferative capacity and multipotency. Their role in astroblastoma formation and growth is unknown.

Abstract

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

Abstract

Bone marrow (BM) cells have recently been shown to give rise to skeletal, hepatic, cardiac, neural, and vascular endothelial tissues. However, it has been shown that this is the result of cell fusion rather than transdifferentiation of hematopoietic stem cells (HSC). For this study, we established a mouse model of brain tumor growth to investigate the differentiation potential of HSC into endothelial cells during brain tumor-induced angiogenesis. Nontransgenic (GFP(neg)) recipient mice were lethally irradiated, and their hematopoietic cells were subsequently repopulated by transplantation of a single green fluorescent protein (GFP)-expressing HSC. Rat glioma (RT-2/RAG) cells were then injected into the striatum of the chimeric mice 6-8 weeks post-transplantation. The animals were sacrificed 3-9 days after tumor implantation, and the mobilization, temporal-spatial distribution, and lineage-specific marker expression profile of the GFP(+) cells within the growing tumor were analyzed. We saw that GFP(+) cells gave rise to elongated, CD34(+)/Flk-1(+) cells that incorporated into the endothelium of tumor blood vessels. However, all GFP(+) cells were also CD45(+), and the presence of CD45 on the HSC-derived endothelial-like cells supports the hypothesis that the hematopoietic cells were recruited into the tumor milieu. The fact that we failed to demonstrate the expression of von Willebrand factor in these cells argues against a true endothelial identity. Nevertheless, the recruitment of HSC-derived endothelial-like cells was an extremely rare event in normal brain parenchyma, and, thus, the permissive influence afforded by the growing tumor appeared to enhance the perivascular tropism and acquisition of an endothelial phenotypes by a population of HSC-derived cells.

Abstract

We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCRbeta gene; express a variety of genes associated with commitment to the T cell lineage, including GATA-3, T cell-specific factor-1, Cbeta, and Id2; and show a surface marker pattern (CD44+ CD25- CD24+ CD5-) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors in the marrow have rearranged the TCR gene loci, express Valpha and Vbeta genes as well as CD3epsilon, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the alphabeta T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN-gamma after stimulation in vitro, and protected the mice against lethal infection with murine CMV. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway in athymic nu/nu mice.

Stem cell research - Paths to cancer therapies and regenerative medicineJAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATIONWeissman, I.2005; 294 (11): 1359-1366

Abstract

Most tissues in complex metazoans contain a rare subset of cells that, at the single-cell level, can self-renew and also give rise to mature daughter cells. Such stem cells likely in development build tissues and are retained in adult life to regenerate them. Cancers and leukemias are apparently not an exception: rare leukemia stem cells and cancer stem cells have been isolated that contain all of the tumorigenicity of the whole tumor, and it is their properties that will guide future therapies. None of this was apparent just 20 years ago, yet this kind of stem cell thinking already provides new perspectives in medical science and could usher in new therapies. Today, political, religious, and ethical issues surround embryonic stem cell and patient-specific pluripotent stem cell research and are center stage in the attempts by governments to ban these fields for discovery and potential therapies. These interventions require physicians and physician-scientists to determine for themselves whether patient welfare or personal ethics will dominate in their practices, and whether all aspects of stem cell research can be pursued in a safe and regulated fashion.

Abstract

The in vivo immunogenicity of Embryonic Stem Cells is controversial. At present, there is only in vitro evidence of MHC I expression by this cell population but vivid speculation about their immune-privileged state. The immunology aspect of ESC transplantation deserves thorough investigation.We injected mouse ESC (expressing Green Fluorescent Protein, GFP) into injured myocardium of syngeneic, allogeneic and SCID recipients. Furthermore, we monitored host response for up to 4 weeks post cell transfer. We determined local response (CD 3, CD 11c expression by host cells), MHC I expression by donor cells, MHC-II expression within and around the graft, humoral response of allogeneic hosts using Flow Cytometry and evaluated the hosts' cytokine response using stimulated spleenocytes by means of ELISPOT. Cell survival was estimated by morphometry, by calculating the area of the GFP+ graft over the area of infarction at multiple sections of the harvested heart.There was significant cellular infiltration into and around the graft consisting of T-lymphocytes (CD3+) and dendritic cells (CD 11c). Infiltration was detectable at 1 week and progressed through 4 weeks following cell transplantation. The humoral Ab response was moderate at 2 weeks but frank at 4 weeks. ELISPOT demonstrated a Th1 pathway of donor specific T-lymphocyte response with strong IFN-gamma and Il-2 production (figure A). MHC I expression was significant within the graft and maximal in the allogeneic groups.An immune response against transplanted ESC was demonstrated and the future use of ESC will likely require the use of systemic immunosuppression.

Abstract

The hematopoietic system is an invaluable model both for understanding basic developmental biology and for developing clinically relevant cell therapies. Using highly purified cells and rigorous microarray analysis we have compared the expression pattern of three of the most primitive hematopoietic subpopulations in adult mouse bone marrow: long-term hematopoietic stem cells (HSC), short-term HSC, and multipotent progenitors. All three populations are capable of differentiating into a spectrum of mature blood cells, but differ in their self-renewal and proliferative capacity. We identified numerous novel potential regulators of HSC self-renewal and proliferation that were differentially expressed between these closely related cell populations. Many of the differentially expressed transcripts fit into pathways and protein complexes not previously identified in HSC, providing evidence for new HSC regulatory units. Extending these observations to the protein level, we demonstrate expression of several of the corresponding proteins, which provide novel surface markers for HSC. We discuss the implications of our findings for HSC biology. In particular, our data suggest that cell-cell and cell-matrix interactions are major regulators of long-term HSC, and that HSC themselves play important roles in regulating their immediate microenvironment.

Identification of mast cell progenitors in adult micePROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChen, C. C., Grimbaldeston, M. A., Tsai, M., Weissman, I. L., Galli, S. J.2005; 102 (32): 11408-11413

Abstract

It is well known that mast cells are derived from hematopoietic stem cells. However, in adult hematopoiesis, a committed mast cell progenitor has not yet been identified in any species, nor is it clear at what point during adult hematopoiesis commitment to the mast cell lineage occurs. We identified a cell population in adult mouse bone marrow, characterized as Lin(-)c-Kit(+)Sca-1(-)-Ly6c(-)FcepsilonRIalpha(-)CD27(-)beta7(+)T1/ST2+, that gives rise only to mast cells in culture and that can reconstitute the mast cell compartment when transferred into c-kit mutant mast cell-deficient mice. In addition, our experiments strongly suggest that these adult mast cell progenitors are derived directly from multipotential progenitors instead of, as previously proposed, common myeloid progenitors or granulocyte/macrophage progenitors.

Abstract

Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly.

Abstract

Growth factors play an essential role in organogenesis. We examine the potential of growth factors to enhance cell engraftment and differentiation and to promote functional improvement after transfer of undifferentiated embryonic stem cells into the injured heart.Green fluorescent protein (GFP)-positive embryonic stem cells derived from 129sv mice were injected into the ischemic area after left anterior descending artery ligation in allogenic (BALB/c) mice. Fifty nanograms of recombinant mouse vascular endothelial growth factor, fibroblast growth factor (FGF), and transforming growth factor (TGF) was added to the cell suspension. Separate control groups were formed in which only the growth factors were given. Echocardiography was performed 2 weeks later to evaluate heart function (fractional shortening [FS]), end-diastolic diameter, and left ventricular wall thickness). Hearts were harvested for histology (connexin 43, alpha-sarcomeric actin, CD3, CD11c, major histocompatability complex class I, hematoxylin-eosin). Degree of restoration (GFP-positive graft/infarct area ratio), expression of cardiac markers, host response, and tumorigenicity were evaluated. Cell transfer resulted in improved cardiac function. TGF-beta led to better restorative effect and a stronger expression of connexin 43, alpha-sarcomeric actin, and major histocompatability complex class I. TGF-beta and FGF retained left ventricular diameter. FS was better in the TGF-beta, FGF, and embryonic stem cells-only group compared with left anterior descending artery-ligated controls. Growth factors with cells (TGF-beta, FGF) resulted in higher FS and smaller end-diastolic diameter than growth factors alone.Growth factors can promote in vivo organ-specific differentiation of early embryonic stem cells and improve myocardial function after cell transfer into an area of ischemic lesion. TGF-beta should be considered as an adjuvant for myocardial restoration with the use of embryonic stem cells.

Abstract

Several studies have reported that bone marrow (BM) cells may give rise to neurons and astrocytes in vitro and in vivo. To further test this hypothesis, we analyzed for incorporation of neural cell types expressing donor markers in normal or injured brains of irradiated mice reconstituted with whole BM or single, purified c-kit(+)Thy1.1(lo)Lin(-)Sca-1(+) (KTLS) hematopoietic stem cells (HSCs), and of unirradiated parabionts with surgically anastomosed vasculature. Each model showed low-level parenchymal engraftment of donor-marker(+) cells with 96-100% immunoreactivity for panhematopoietic (CD45) or microglial (Iba1 or Mac1) lineage markers in all cases studied. Other than one arborizing structure in the olfactory bulb of one BM-transplanted animal, possibly representing a neuronal or glial cell process, we found no donor-marker-expressing astrocytes or non-Purkinje neurons among >10,000 donor-marker(+) cells from 21 animals. These data strongly suggest that HSCs and their progeny maintain lineage fidelity in the brain and do not adopt neural cell fates with any measurable frequency.

Abstract

The binding of frizzled (Fzd) receptors by their Wnt ligands results in the inhibition of beta-catenin degradation and subsequent transcription of beta-catenin/LEF-inducible genes. The beta-catenin pathway is known to be involved in development, tumorigenesis, and stem cell self-renewal. In humans, the FZD9 gene lies in the region of chromosome 7q11.23 deleted in the neurodevelopmental disorder, Williams-Beuren syndrome (WBS). Fzd9-/- mice show no obvious features of WBS, but reveal a role for Fzd9 in lymphoid development and maturation. Fzd9-/- mice show pronounced splenomegaly, thymic atrophy, and lymphadenopathy with age, with accumulation of plasma cells in lymph nodes. There is a depletion of developing B cells in the bone marrow (BM), particularly in the pre-B stage where immunoglobulin heavy chains are expressed and the cells are undergoing clonal expansion prior to light chain rearrangement. The pre-B defect is partially intrinsic to the hematopoietic system; as in competitive BM reconstitution studies, Fzd9-/- -derived BM exhibits defective B-cell development when implanted into a wild-type host. Mature B cells are present in normal numbers in lymph node and spleen. These findings suggest a role for Fzd9 signaling in lymphoid development, particularly at points where B cells undergo self-renewal prior to further differentiation.

Abstract

Leukemia inhibitory factor (LIF) is required, but not sufficient, for pluripotent mouse embryonic stem (ES) cell expansion in vitro in the absence of serum or a feeder cell layer, suggesting that additional signals are provided by serum or feeders that are necessary to support self-renewal. Here we show that transgenic ES cell lines expressing Bcl-2, an antiapoptotic protein, continue to self-renew in serum- and feeder-free conditions when supplemented with LIF; even in the absence of bone morphogenic proteins. Bcl-2-expressing clones sustain the characteristics of undifferentiated, pluripotent ES cells during long-term culture, and maintain their potential to differentiate into mature cell types. These results suggest that LIF and Bcl-2 overexpression are sufficient to expand these mouse pluripotent stem cells in vitro.

Abstract

The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-alpha and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-alpha complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.

Abstract

The aim of this study was to determine the lineage origin of interferon-alpha-producing cells (IPCs), also called plasmacytoid dendritic cells, in mice by evaluating the ability of common lymphoid (CLP) and myeloid (CMP) progenitors to give rise to IPCs.Sublethally irradiated C57Bl/6 mice were intravenously transplanted with rigorously purified lymphoid and myeloid progenitors from a congenic mouse strain. At various time points posttransplantation mice were analyzed for donor-derived cells by flow cytometry. The developmental potential of all progenitor populations was also tested in in vitro cultures. In addition, in vitro and in vivo derived IPCs were functionally assessed for their interferon-alpha production after virus challenge.Transplantation of 1 x 10(4) common myeloid progenitors, 1 x 10(4) common lymphoid progenitors or 2.5 x 10(4) granulocyte/macrophage progenitors all led to the generation of IPCs within 2 to 3 weeks. In general, IPC reconstitution in spleen and liver by CMPs was more efficient than by CLP. Adding Flt3L alone to in vitro cultures was sufficient to support the development of IPCs from myeloid progenitors whereas CLPs required additional survival factors provided either by stroma cells or by introduction of transgenic Bcl-2. Both myeloid- and lymphoid-derived IPC were indistinguishable by function, gene expression, and morphology.Surprisingly, our results clearly show that murine IPCs differentiate from both lineages but are mainly of myeloid origin. These results extend to IPCs the observation made originally in classical dendritic cells that cellular expression of so called lineage markers does not correlate with lineal origin.

Abstract

Stem cells are cells that at the single cell level both self-renew and give rise to differentiated progeny. Self renewal is the property that distinguishes stem cells and progenitors, and in the blood-forming system explains why haematopoietic stem cells (HSCs), not progenitors, are the only cells capable of providing rapid and sustained regeneration of the blood-forming system after ablation by cancer chemo- and radiotherapies. Cancer-free prospectively purified HSCs regenerate the haematopoietic system of patients as rapidly as a marrow or mobilized blood transplant, but without the risk of re-seeding the body with cancer cells. Further, purified allogeneic HSCs can establish donor-specific tolerance to subsequent tissue grafts. However, in contrast to widely-publicized reports of HSC plasticity, we have not been able to show transdifferentiation of HSC to muscle, heart, brain or gut, and conclude that rare cell fusions and incomplete purifications are likely explanations for the other published results. The ability to self-renew is also potentially dangerous, as poorly regulated self renewal is, we believe, a central lesion in all cancers. We have recently shown that myeloid leukaemias in mouse and human are often driven by rare leukaemia (cancer) stem cells which are at the progenitor stage of differentiation, but have activated the self-renewing cell division pathway normally used only by HSCs. Similar cancer stem cells have been isolated in other tumours.

Abstract

A vast literature exists on the biology of blood formation and regeneration under experimental and clinical conditions. The field of hematopoiesis was recently advanced by the capacity to purify to homogeneity primitive hematopoietic stem and progenitor cells. Isolation of cells at defined maturational stages has enhanced the understanding of the fundamental nature of stem cells, including how cell fate decisions are made, and this understanding is relevant to the development of other normal as well as malignant tissues. This review updates the basic biology of hematopoietic stem cells (HSC) and progenitors, the evolving use of purified HSC as grafts for clinical hematopoietic cell transplantation (HCT) including immune tolerance induction, and the application of HSC biology to other stem cell fields.

Abstract

Leukemia stem cells are defined as transformed hematopoietic stem cells or committed progenitor cells that have amplified or acquired the stem cell capacity for self-renewal, albeit in a poorly regulated fashion. In this issue of Cancer Cell, Huntly and colleagues report a striking difference in the ability of two leukemia-associated fusion proteins, MOZ-TIF2 and BCR-ABL, to transform myeloid progenitor populations. This rigorous study supports the idea of a hierarchy among leukemia-associated protooncogenes for their ability to endow committed myeloid progenitors with the self-renewal capacity driving leukemic stem cell propagation, and sheds new light on the pathogenesis of chronic and acute myelogenous leukemias.

Abstract

We previously described the generation of non-obese diabetic (NOD) mice expressing a transgenic T cell receptor (TCR) specific for peptide epitope 286-300 of the diabetes related self antigen, glutamic acid decarboxylase (GAD)65 in the context of I-A(g7) class II MHC, that are paradoxically protected from diabetes. In this report, we examine the atypical CD8+ cells in these mice. Unlike typical class II restricted TCR transgenic mice, GAD286 mice have normal numbers of CD8+ cells, half of which express high levels of the transgenic TCR. These MHC mismatched CD8+ cells persist in the periphery and proliferate to GAD286-300 peptide in vitro and in vivo in a class II restricted fashion. Interestingly, the CD8+ tetramer(-) T cells that are expressing endogenous TCR can delay diabetes induction in a transfer model, as we previously showed for CD4+ tetramer+ T cells in these mice. The MHC mismatched CD8+ cells appear to be positively selected in an atypical fashion, in that they do not upregulate CD69 or reexpress CD44, and they escape negative selection. We find that production of these CD8+ cells is not dependent on NOD thymus or high affinity of the TCR, but is dependent on the atypical TCR transgenic thymic environment.

Abstract

Neovascularization is essential for tumor growth and invasion. Mounting evidence suggests that tumor cells recruit circulating endothelial progenitor cells to promote vasculogenesis to compliment tumor angiogenesis. This study examines the constitutive role of bone marrow-derived cells in this process.Rat glioma cells were implanted into brains of T-cell-depleted knockout mice. At various timepoints after tumor implantation, naïve bone marrow cells from ubiquitous transgenic mice expressing green fluorescent protein (GFP) were infused into these animals. The incorporation of GFP-positive cells into the vascular architecture was visualized by fluorescence confocal microscopy in conjunction with the transcription profiles of vascular endothelial growth factor (VEGF) and angiopoietin-1 and -2 (Ang-1 and Ang-2).Of the cells infused, 8 days after tumor implantation, 0.49% were found exclusively sequestered in the vicinity of tumor vessels. This coincided with a decline in the expression of Ang-1 and a rise in the expression of VEGF and Ang-2. A few of these cells (0.66 of the 0.49%) localized onto the vascular wall. They resembled endothelial cells and expressed vWF.The incorporation of bone marrow-derived unpurified endothelial cells into the tumor vascular bed is both time-limited and infrequent. These cells may play a supportive rather than a constitutive role in tumor neovascularization.

Abstract

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

Abstract

The AP-1 transcription factor JunB is a transcriptional regulator of myelopoiesis. Inactivation of JunB in postnatal mice results in a myeloproliferative disorder (MPD) resembling early human chronic myelogenous leukemia (CML). Here, we show that JunB regulates the numbers of hematopoietic stem cells (HSC). JunB overexpression decreases the frequency of long-term HSC (LT-HSC), while JunB inactivation specifically expands the numbers of LT-HSC and granulocyte/macrophage progenitors (GMP) resulting in chronic MPD. Further, we demonstrate that junB inactivation must take place in LT-HSC, and not at later stages of myelopoiesis, to induce MPD and that only junB-deficient LT-HSC are capable of transplanting the MPD to recipient mice. These results demonstrate a stem cell-specific role for JunB in normal and leukemic hematopoiesis and provide experimental evidence that leukemic stem cells (LSC) can reside at the LT-HSC stage of development in a mouse model of MPD.

Abstract

Telomerase is critical for the protection of germ line and stem cell chromosomes from fatal shortening during replication. In most organisms, telomerase activity is suppressed in progressively committed cells and falls to basal rates in terminally differentiated lineages. The colonial ascidian Botryllus schlosseri propagates asexually and sexually, presumably from pools of stem cells that self-renew throughout the 2- to 5-year colony life span. Asexual budding takes place continuously from the parental body wall. When the colony reaches a critical size, sexual reproduction commences with the generation of gonads. Here, we establish the existence of 6-15 kb telomeres on the ends of Botryllus chromosomes. We develop a real-time quantitative PCR telomeric repeat amplification protocol (TRAP) assay that reliably detects 0.2-100 TPG units in cells and tissues. We find highest levels of enzymatic activity in the gonads, developing embryos, and tissues containing the earliest asexual buds. Telomerase activity appears to be suppressed in later buds during organogenesis and falls to basal rates in mature zooids. We postulate that this pattern reflects maximum telomere restoration in somatic stem cells of early buds and suppression of telomerase activity in progenitors and terminally differentiated cells, indicative of an alternate role for stem cells as repeated body regenerators in colonial life histories.

Abstract

The progression of chronic myelogenous leukemia (CML) to blast crisis is supported by self-renewing leukemic stem cells. In normal mouse hematopoietic stem cells, the process of self-renewal involves the beta-catenin-signaling pathway. We investigated whether leukemic stem cells in CML also use the beta-catenin pathway for self-renewal.We used fluorescence-activated cell sorting to isolate hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors from marrow during several phases of CML and from normal marrow. BCR-ABL, beta-catenin, and LEF-1 transcripts were compared by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay in normal and CML hematopoietic stem cells and granulocyte-macrophage progenitors. Confocal fluorescence microscopy and a lymphoid enhancer factor/T-cell factor reporter assay were used to detect nuclear beta-catenin in these cells. In vitro replating assays were used to identify self-renewing cells as candidate leukemic stem cells, and the dependence of self-renewal on beta-catenin activation was tested by lentiviral transduction of hematopoietic progenitors with axin, an inhibitor of the beta-catenin pathway.The granulocyte-macrophage progenitor pool from patients with CML in blast crisis and imatinib-resistant CML was expanded, expressed BCR-ABL, and had elevated levels of nuclear beta-catenin as compared with the levels in progenitors from normal marrow. Unlike normal granulocyte-macrophage progenitors, CML granulocyte-macrophage progenitors formed self-renewing, replatable myeloid colonies, and in vitro self-renewal capacity was reduced by enforced expression of axin.Activation of beta-catenin in CML granulocyte-macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells.

Abstract

We characterize the survival, migration, and differentiation of human neurospheres derived from CNS stem cells transplanted into the ischemic cortex of rats 7 days after distal middle cerebral artery occlusion. Transplanted neurospheres survived robustly in naive and ischemic brains 4 wk posttransplant. Survival was influenced by proximity of the graft to the stroke lesion and was negatively correlated with the number of IB4-positive inflammatory cells. Targeted migration of the human cells was seen in ischemic animals, with many human cells migrating long distances ( approximately 1.2 mm) predominantly toward the lesion; in naive rats, cells migrated radially from the injection site in smaller number and over shorter distances (0.2 mm). The majority of migrating cells in ischemic rats had a neuronal phenotype. Migrating cells between the graft and the lesion expressed the neuroblast marker doublecortin, whereas human cells at the lesion border expressed the immature neuronal marker beta-tubulin, although a small percentage of cells at the lesion border also expressed glial fibrillary acid protein (GFAP). Thus, transplanted human CNS (hCNS)-derived neurospheres survived robustly in naive and ischemic brains, and the microenvironment influenced their migration and fate.

Abstract

Varicella-zoster virus (VZV) establishes latency in sensory ganglia and causes herpes zoster upon reactivation. These investigations in a nonobese diabetic severe combined immunodeficient mouse-human neural cell model showed that VZV infected both neurons and glial cells and spread efficiently from cell to cell in vivo. Neural cell morphology and protein synthesis were preserved, in contrast to destruction of epithelial cells by VZV. Expression of VZV genes in neural cells was characterized by nuclear retention of the major viral transactivating protein and a block in synthesis of the predominant envelope glycoprotein. The attenuated VZV vaccine strain retained infectivity for neurons and glial cells in vivo. VZV gene expression in differentiated human neural cells in vivo differs from neural infection by herpes simplex virus, which is characterized by latency-associated transcripts, and from lytic VZV replication in skin. The chimeric nonobese diabetic severe combined immunodeficient mouse model may be useful for investigating other neurotropic human viruses.

Abstract

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.

Abstract

Differential expression of Hox genes is associated with normal hematopoiesis, whereas inappropriate maintenance of Hox gene expression, particularly Hoxa7 and Hoxa9, is a feature of leukemias harboring mixed-lineage leukemia (MLL) mutations. To understand the pathogenic roles of Hox genes in MLL leukemias, we assessed the impact of Hoxa7 or Hoxa9 nullizygosity on hematopoietic progenitor compartments and their susceptibility to MLL-induced leukemias. Selective reductions in the absolute numbers of committed progenitors, but not of hematopoietic stem cells, distinguished Hoxa7- and Hoxa9-deficient mice. Megakaryocytic/erythroid progenitor (MEP) reductions in Hoxa7(-/-) mice correlated with reticulocytosis and thrombocytopenia without anemia. Conversely, Hoxa9(-/-) mice displayed marked lymphopenia and substantial reductions of common lymphoid progenitors (CLPs) and lymphoid precursors, in addition to significant reductions of common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs). In retroviral transduction/transplantation assays, Hoxa7- and Hoxa9-deficient progenitors remained susceptible to transformation by MLL-GAS7, which activates MLL through a dimerization-dependent mechanism. However, Hoxa7(-/-) or Hoxa9(-/-) progenitors were less efficient in generating transformed blast colony-forming units (CFUs) in vitro and induced leukemias with longer disease latencies, reduced penetrance, and less mature phenotypes. Thus, Hoxa7 and Hoxa9 contribute to hematopoietic progenitor homeostasis but are not necessary for MLL-GAS7-mediated leukemogenesis, yet they appear to affect disease latency, penetrance, and phenotypes consistent with their critical roles as downstream targets of MLL fusion proteins.

Abstract

Under conditions of tissue injury, myocardial replication and regeneration have been reported. A growing number of investigators have implicated adult bone marrow (BM) in this process, suggesting that marrow serves as a reservoir for cardiac precursor cells. It remains unclear which BM cell(s) can contribute to myocardium, and whether they do so by transdifferentiation or cell fusion. Here, we studied the ability of c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells to regenerate myocardium in an infarct model. Cells were isolated from transgenic mice expressing green fluorescent protein (GFP) and injected directly into ischaemic myocardium of wild-type mice. Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable. These GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45 and myeloid marker Gr-1. We also studied the role of circulating cells in the repair of ischaemic myocardium using GFP+-GFP- parabiotic mice. Again, we found no evidence of myocardial regeneration from blood-borne partner-derived cells. Our data suggest that even in the microenvironment of the injured heart, c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells adopt only traditional haematopoietic fates.

Abstract

Recent years have seen much excitement over the possibility that adult mammalian stem cells may be capable of differentiating across tissue lineage boundaries, and as such may represent novel, accessible, and very versatile effectors of therapeutic tissue regeneration. Yet studies proposing such "plasticity" of adult somatic stem cells remain controversial, and in general, existing evidence suggests that in vivo such unexpected transformations are exceedingly rare and in some cases can be accounted for by equally unexpected alternative explanations.

Abstract

The major site of hematopoiesis transitions from the fetal liver to the spleen and bone marrow late in fetal development. To date, experiments have not been performed to evaluate functionally the migration and seeding of hematopoietic stem cells (HSCs) during this period in ontogeny. It has been proposed that developmentally timed waves of HSCs enter the bloodstream only during distinct windows to seed the newly forming hematopoietic organs. Using competitive reconstitution assays to measure HSC activity, we determined the localization of HSCs in the mid-to-late gestation fetus. We found that multilineage reconstituting HSCs are present at low numbers in the blood at all timepoints measured. Seeding of fetal bone marrow and spleen occurred over several days, possibly while stem cell niches formed. In addition, using dual-chamber migration assays, we determined that like bone marrow HSCs, fetal liver HSCs migrate in response to stromal cell-derived factor-1alpha (SDF-1alpha); however, unlike bone marrow HSCs, the migratory response of fetal liver HSCs to SDF-1alpha is greatly increased in the presence of Steel factor (SLF), suggesting an important role for SLF in HSC homing to and seeding of the fetal hematopoietic tissues. Together, these data demonstrate that seeding of fetal organs by fetal liver HSCs does not require large fluxes of HSCs entering the fetal bloodstream, and that HSCs constitutively circulate at low levels during the gestational period from 12 to 17 days postconception. Newly forming hematopoietic tissues are seeded gradually by HSCs, suggesting initial seeding is occurring as hematopoietic niches in the spleen and bone marrow form and become capable of supporting HSC self-renewal. We demonstrate that fetal and adult HSCs exhibit specific differences in chemotactic behavior. While both migrate in response to SDF-1alpha, fetal HSCs also respond significantly to the cytokine SLF. In addition, the combination of SDF-1alpha and SLF results in substantially enhanced migration of fetal HSCs, leading to migration of nearly all fetal HSCs in this assay. This finding indicates the importance of the combined effects of SLF and SDF-1alpha in the migration of fetal HSCs, and is, to our knowledge, the first demonstration of a synergistic effect of two chemoattractive agents on HSCs.

Abstract

Colonial organisms provide a unique experimental system for stem cell biology. The colonial Urochordate Botryllus schlosseri reproduces sexually as well as by continuous asexual budding. Adjacent colonies with a shared histocompatibility allele undergo vascular fusion and establish a common blood circulation, performing natural transplantation. Fused colonies become chimeras, often with complete somatic replacement of the host cell genotype by the fused parabiont. We attempted to establish a radioprotection assay for the somatic stem cells that induce long-term chimerism in Botryllus. We demonstrate over a range of radiation doses that neither autologous nor allogeneic cell transplantation enhances survival of host colonies. This suggests that high mitotic index associated with continuous asexual development leads to radiosensitivity of organs and structures essential to survival during engraftment. We observe that radiation induces uncontrolled epithelial cell proliferation in abnormally terminated buds, suggesting that stem cells are not required for the initial stages of bud development.

Abstract

Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. In addition, dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve.

Abstract

To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion.

Abstract

Insulin-like growth factor-1 (IGF-1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF-1 might enhance cellular engraftment and host organ-specific differentiation after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF-1 to the injected cell population. Green fluorescent protein (GFP)-labeled sv129 ESCs (2.5 x 10(5)) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF-1 (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF-1 (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF-1 group (p = .025). There was a trend toward higher fractional shortening in the IGF-treated group. Histological analysis demonstrated that IGF-1 promoted expression of alpha-sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF-1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

Abstract

To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c-kit+ Thy1.1lo Lin- Sca-1+ hematopoietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker-expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor-derived myofibers expressed both donor-specific and host-specific markers, suggesting that they arise by low-level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage.

Abstract

We have used the hematopoietic system as a model to investigate whether acute myeloid leukemia arises exclusively from self-renewing stem cells or also from short-lived myeloid progenitors. When transduced with a leukemogenic MLL fusion gene, prospectively isolated stem cells and myeloid progenitor populations with granulocyte/macrophage differentiation potential are efficiently immortalized in vitro and result in the rapid onset of acute myeloid leukemia with similar latencies following transplantation in vivo. Regardless of initiating cell, leukemias displayed immunophenotypes and gene expression profiles characteristic of maturation arrest at an identical late stage of myelomonocytic differentiation, putatively a monopotent monocytic progenitor stage. Our findings unequivocally establish the ability of transient repopulating progenitors to initiate myeloid leukemias in response to an MLL oncogene, and support the existence of cancer stem cells that do not necessarily overlap with multipotent stem cells of the tissue of origin.

Abstract

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.

Abstract

The colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction which is controlled by a single, highly polymorphic locus called the Fu/HC. We are using map-based cloning to identify Fu/HC gene(s), and have currently delineated their location to an approximately 1-cM region of the B. schlosseri genome. The Fu/HC physical map currently consists of 85 sequence-tagged sites mapped on a minimum tiling path of 800 kb which consists of five contigs, with four gaps remaining to be crossed, and is estimated to be 75% completed. Approximately half this region has been sequenced throughout the locus, allowing the first analysis of a metazoan histocompatibility locus outside of vertebrates. This has resulted in the identification of 18 predicted genes, a number of which have been found to be expressed. Several of these genes are well conserved among the chordates; however, none of the predicted or expressed genes are linked within the genome of any organism in the databases. In addition, the Fu/HC is one of the most polymorphic loci ever described, and physical mapping has revealed that the locus is quite dynamic, and includes features such as hotspots of recombination.

Abstract

The formation of chimeric colonies following allogeneic contact between benthic invertebrates may strongly affect colony fitness. Here we show that, in a field population of the colonial ascidian Botryllus schlosseri in Monterey Bay, California, more than 20% of all colonies occur in allogeneic contact with conspecifics. We experimentally assessed the effects of allogeneic contact on the following life-history traits under natural field conditions: growth, age and size at first reproduction, and egg production (fecundity). When compared with isolated colonies, and in some cohorts also with colonies that rejected allogeneic neighbors, colonies that fused with neighbors incurred reduced fitness in terms of most life-history traits measured. We propose that one of the benefits of precise allorecognition is that, in fused colonies, it limits the unit of selection to chimeric individuals composed of closely related kin.

Abstract

Leukemia can be viewed as a newly formed, abnormal hematopoietic tissue initiated by a few leukemic stem cells (LSCs) that undergo an aberrant and poorly regulated process of organogenesis analogous to that of normal hematopoietic stem cells. A hallmark of all cancers is the capacity for unlimited self-renewal, which is also a defining characteristic of normal stem cells. Given this shared attribute, it has been proposed that leukemias may be initiated by transforming events that take place in hematopoietic stem cells. Alternatively, leukemias may also arise from more committed progenitors caused by mutations and/or selective expression of genes that enhance their otherwise limited self-renewal capabilities. Identifying the LSCs for each type of leukemia is a current challenge and a critical step in understanding their respective biologies and may provide key insights into more effective treatments. Moreover, LSC identification and purification will provide a powerful diagnostic, prognostic, and therapeutic tool in the clinic.

Abstract

Transgenic mice that over-express B cell leukemia/lymphomas (Bcl)-2 in myeloid cells under control of the human MRP8 promoter (hMRP8-Bcl-2) or in T lymphocytes under the E micro promoter (E micro -Bcl-2) were compared with C57BL/6 control mice following cecal ligation and puncture (CLP). There was a significant difference in outcome between the hMRP8-Bcl-2 and control mice with 100% survival in the hMRP8-Bcl-2 mice vs 25% survival in the control mice. In separate experiments there was a significant difference between E micro -Bcl-2 and control mice with 87.5 and 22.2% survival, respectively. Adoptive transfer of CD11b-positive bone marrow cells from hMRP8-Bcl-2 or C57BL/6 mice to C57BL/6 mice subjected to CLP resulted in 100 and 0% survival, respectively. Adoptive transfer of CD11b-positive cells from either hMRP8-Bcl-2 or C57BL/6 mice to Rag-1(-/-) mice (no mature T or B cells) subjected to CLP resulted in survival of 87.5 and 12.5%, respectively. The hMRP8-Bcl-2 mice had significantly more neutrophils and fewer bacteria in the peritoneum compared with C57BL/6 mice 24 h after CLP. These experiments show that Bcl-2 over-expression is protective in CLP and that protection is independent of lymphocytes. We propose that over-expression of Bcl-2 in T cells or myeloid cells induce release of a molecule(s) that protects against death following CLP.

Expression of BCR/ABL and BCL-2 in myeloid progenitors leads to myeloid leukemiasPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAJaiswal, S., Traver, D., Miyamoto, T., Akashi, K., Lagasse, E., Weissman, I. L.2003; 100 (17): 10002-10007

Abstract

Chronic myelogenous leukemia is a myeloproliferative disorder (MPD) that, over time, progresses to acute leukemia. Both processes are closely associated with the t(9;22) chromosomal translocation that creates the BCR/ABL fusion gene in hematopoietic stem cells (HSCs) and their progeny. Chronic myelogenous leukemia is therefore classified as an HSC disorder in which a clone of multipotent HSCs is likely to be malignantly transformed, although direct evidence for malignant t(9;22)+ HSCs is lacking. To test whether HSC malignancy is required, we generated hMRP8p210BCR/ABL transgenic mice in which expression of BCR/ABL is absent in HSCs and targeted exclusively to myeloid progenitors and their myelomonocytic progeny. Four of 13 BCR/ABL transgenic founders developed a chronic MPD, but only one progressed to blast crisis. To address whether additional oncogenic events are required for progression to acute disease, we crossed hMRP8p210BCR/ABL mice to apoptosis-resistant hMRP8BCL-2 mice. Of 18 double-transgenic animals, 9 developed acute myeloid leukemias that were transplantable to wild-type recipients. Taken together, these data indicate that a MPD can arise in mice without expression of BCR/ABL in HSCs and that additional mutations inhibiting programmed cell death may be critical in the transition of this disease to blast-crisis leukemia.

Abstract

nu/nu mice fail to develop a thymus and mature T cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. We investigated whether early T cell progenitor development in the nu/nu bone marrow is also defective. We demonstrated a maturation arrest of nu/nu marrow T cell progenitors associated with a lack of pTalpha gene expression and a failure to give rise to mature T cells in adoptive euthymic hosts. Wild-type hemopoietic stem cells rapidly matured into functional T cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. We show that defects in bone marrow prethymic T cell development can also contribute to T cell deficiency in nu/nu mice.

Abstract

Stimulation of Flt3 receptor tyrosine kinase through its cognate ligand expands early hematopoietic progenitor and dendritic cells (DCs) in humans and mice. The exact developmental stages at which hematopoietic progenitors express Flt3, are responsive to its ligand, and subsequently develop to DCs, are not known. Here we show that common lymphoid and common myeloid progenitors, as well as steady state DCs in thymus, spleen, and epidermis, express Flt3. The receptor is down-regulated once definitive B cell, T cell, and megakaryocyte/erythrocyte commitment occurs, and Flt3 is not detectable on other steady state hematopoietic cell populations. Upon in vivo Flt3 ligand (Flt3L) administration, Flt3+ progenitor cells and their progeny DCs are expanded, whereas Flt3- downstream progenitors are not, or are only slightly increased. Transplantation of common lymphoid and common myeloid progenitors and subsequent Flt3L injection increases progeny DCs of both precursor populations. These findings provide a definitive map of Flt3 expression in the hematopoietic hierarchy and directly demonstrate that Flt3L can drive DC development along both the lymphoid and myeloid developmental pathways from Flt3+ progenitors to Flt3+ DCs.

Abstract

Telomere shortening ultimately limits the replicative life span of cultured human somatic cells. Telomeres also shorten during replicative aging in vivo in hematopoietic cells, including early hematopoietic progenitors and hematopoietic stem cells (HSCs), from humans and mice, despite readily detectable levels of telomerase in these cells. To assess the relevance of telomerase to the long-term replicative capacity of HSCs in vivo, we serially transplanted HSCs from wild-type and telomerase-deficient mice until exhaustion and monitored telomere length in HSCs during this process. Telomerase-deficient HSCs could be serially transplanted for only 2 rounds, whereas wild-type HSCs could be serially transplanted for at least 4 rounds. Furthermore, the rate of telomere shortening was increased approximately 2-fold during serial transplantation of telomerase-deficient HSCs. These findings suggest that one role for telomerase in the HSC is to partially counter the rate of telomere shortening during division of HSCs, thereby preventing premature loss of telomere function and providing added replicative capacity.

Abstract

Lifelong self-renewal is a unique property of somatic stem cells. Recently, several primitive multipotent yet committed (non-self-renewing) hematopoietic progenitor populations were identified in mouse bone marrow. We have characterized the expression of 1200 selected mouse genes using the Atlas cDNA array in highly purified hematopoietic stem cells (HSCs) and 6 closely related progenitor populations: common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythrocyte progenitors (MEPs), common lymphoid progenitors (CLPs), and pro-T and pro-B cells. Cluster analysis revealed that nearly half of all differentially expressed transcripts are associated with HSCs, supporting the notion of an active transcriptional status of HSCs. Genes found enriched in the HSC cluster encompass many developmentally regulated genes, some previously associated with HSC self-renewal. In contrast, genes that are enriched in committed progenitors are mostly associated with hematopoietic differentiation, immune regulation, and metabolism. Thus, the transition from HSCs toward committed progenitors correlates with the down-regulation of a large number of HSC-associated genes and progressive up-regulation of a limited number of lineage-specific genes. These genetic analyses revealed both quantitative and qualitative differences between the transcripts associated with HSCs versus downstream progenitors and produced a list of the candidate genes, potentially involved in HSC self-renewal.

Abstract

Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro.Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents.Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

Abstract

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.

Abstract

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Abstract

A central issue in stem cell biology is to understand the mechanisms that regulate the self-renewal of haematopoietic stem cells (HSCs), which are required for haematopoiesis to persist for the lifetime of the animal. We found that adult and fetal mouse and adult human HSCs express the proto-oncogene Bmi-1. The number of HSCs in the fetal liver of Bmi-1-/- mice was normal. In postnatal Bmi-1-/- mice, the number of HSCs was markedly reduced. Transplanted fetal liver and bone marrow cells obtained from Bmi-1-/- mice were able to contribute only transiently to haematopoiesis. There was no detectable self-renewal of adult HSCs, indicating a cell autonomous defect in Bmi-1-/- mice. A gene expression analysis revealed that the expression of stem cell associated genes, cell survival genes, transcription factors, and genes modulating proliferation including p16Ink4a and p19Arf was altered in bone marrow cells of the Bmi-1-/- mice. Expression of p16Ink4a and p19Arf in normal HSCs resulted in proliferative arrest and p53-dependent cell death, respectively. Our results indicate that Bmi-1 is essential for the generation of self-renewing adult HSCs.

Abstract

The antigen receptors on cells of innate immune systems recognize broadly expressed markers on non-host cells while the receptors on lymphocytes of the adaptive immune system display a higher level of specificity. Adaptive immunity, with its exquisite specificity and immunological memory, has only been found in the jawed vertebrates, which also display innate immunity. Jawless fishes and invertebrates only have innate immunity. In the adaptive immune response, T and B-lymphocytes detect foreign agents or antigens using T cell receptors (TCR) or immunoglobulins (Ig), respectively. While Ig can bind free intact antigens, TCR only binds processed antigenic fragments that are presented on molecules encoded in the major histocompatibility complex (MHC). MHC molecules display variation through allelic polymorphism. A diverse repertoire of Ig and TCR molecules is generated by gene rearrangement and junctional diversity, processes carried out by the recombinase activating gene (RAG) products and terminal deoxynucleotidyl transferase (TdT). Thus, the molecules that define adaptive immunity are TCR, Ig, MHC molecules, RAG products and TdT. No direct predecessors of these molecules have been found in the jawless fishes or invertebrates. In contrast, the complement cascade can be activated by either adaptive or innate immune systems and contains examples of molecules that gradually evolved from non-immune functions to being part of the innate and then adaptive immune system. In this paper we examine the molecules of the adaptive immune system and speculate on the existence of direct predecessors that were part of innate immunity.

Abstract

The colonial protochordate Botryllus schlosseri is genetically manipulable and represents a potential model organism for a variety of biological disciplines, including immunology, stem cell biology and development. This article presents the construction and characterization of both BAC and fosmid genomic libraries of the 725-Mbp B. schlosseri genome. The BAC library currently consists of 2x genome coverage with an average insert size of 80 kb. The fosmid library is at 11x genome coverage with an average insert of 40 kb. B. schlosseri is a small organism containing a large number of compounds that hinder DNA purification. Thus a number of protocols had to be modified in order to make purified, high molecular weight inserts for cloning, including both gel purification and insert concentration techniques. Both libraries were characterized by using them in initial physical mapping of a single histocompatibility locus, and were found to be representative and functional. These libraries are important tools for physical mapping and positional cloning in the B. schlosseri genome, and the techniques adapted to make them are suitable for use on other organisms in which high molecular weight DNA is difficult to purify.

Abstract

A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes.

Characterization of mouse clonogenic megakaryocyte progenitorsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICANakorn, T. N., Miyamoto, T., Weissman, I. L.2003; 100 (1): 205-210

Abstract

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes, with a plating efficiency >60% at the single-cell level. In vivo, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.

Abstract

Purified hematopoietic stem cells (HSCs) were transplanted into NOD mice to test whether development of hyperglycemia could be prevented. Engraftment of major histocompatibility complex-mismatched HSCs was compared with bone marrow (BM) grafts. HSCs differed from BM because HSCs were more strongly resisted and HSC recipients retained significant levels of NOD T-cells, whereas BM recipients were full donor chimeras. Despite persistent NOD T-cells, all HSC chimeras were protected from hyperglycemia, and attenuation of islet lesions was observed. T-cell selection was altered in allogeneic HSC recipients as demonstrated by deletion of both donor and host superantigen-specific T-cells. Syngeneic and congenic hematopoietic cell transplants were also performed to differentiate the influence of the preparative regimen(s) versus the allografts. Unlike the allogeneic HSC transplantations, syngeneic or congenic grafts did not retard diabetes development. In a pilot study, overtly diabetic NOD mice were cured by co-transplantation of allogeneic HSCs and donor-matched islets. We conclude that allogeneic HSC transplants block allo- and autoimmunity, despite residual host T-cell presence. These data demonstrate for the first time that purified HSC grafts block development of autoimmune diabetes and illuminate how HSC grafts alter thymic and peripheral T-cell responses against auto- and alloantigens.

Abstract

Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases.

Abstract

Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections. In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa. Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP. Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen. A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival. Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase. Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection. These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

Abstract

Telomeres shorten in hematopoietic cells, including hematopoietic stem cells (HSCs), during aging and after transplantation, despite the presence of readily detectable levels of telomerase in these cells. In T cells, antigenic stimulation has been shown to result in a marked increase in the level of telomerase activity. We now show that stimulation of T cells derived from serially transplanted HSC results in a telomerase-dependent elongation of telomere length to a size similar to that observed in T cells isolated directly from young mice. Southern analysis of telomere length in resting and anti-CD3/CD28 stimulated donor-derived splenic T cells revealed an increase in telomere size by approximately 7 kb for the population as a whole. Stimulation of donor-derived T cells from recipients of HSCs from telomerase-deficient mice did not result in regeneration of telomere length, demonstrating a dependence on telomerase. Furthermore, clonal anti-CD3/CD28 stimulation of donor-derived T cells followed by fluorescent in situ hybridization (FISH) analysis of telomeric signal intensity showed that telomeres had increased in size by approximately 50% for all clonal expansions. Together, these results imply that one role for telomerase in T cells may be to renew or extend replicative potential via the rejuvenation of telomere length.

Abstract

The identification of the common lymphoid progenitors in mouse bone marrow allows us to directly assess the regulatory mechanisms of lymphoid lineage commitment. The unexpected finding of a latent myeloid differentiation potential in lymphoid progenitors sheds light on the importance of cytokine receptor expression at this stage. We will discuss the biological nature of common lymphoid progenitors as a model of differentiation from multipotent to lineage committed progenitors. Elucidation of this hidden differentiation potential in progenitors will help further our understanding of the molecular mechanisms that control the cell fate determination of not only common lymphoid progenitors, but also their ancestors, hematopoietic stem cells, and their descendents such as committed T and B cell progenitors.

Abstract

Langerhans cells (LCs) are bone marrow (BM)-derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors.

Abstract

IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.

Abstract

To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GFP+:GFP- parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all.

Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: A model for development and regenerationDEVELOPMENTAL BIOLOGYLauzon, R. J., Ishizuka, K. J., Weissman, I. L.2002; 249 (2): 333-348

Abstract

Botryllus schlosseri is a colonial marine urochordate in which all adult organisms (called zooids) in a colony die synchronously by apoptosis (programmed cell death) in cyclical fashion. During this death phase called takeover, cell corpses within the dying organism are engulfed by circulating phagocytic cells. The "old" zooids and their organs are resorbed within 24-36 h (programmed cell removal). This process coincides temporally with the growth of asexually derived primary buds, that harbor a small number of undifferentiated cells, into mature zooids containing functional organs and tissues with the same body plan as adult zooids from which they budded. Within these colonies, all zooids share a ramifying network of extracorporeal blood vessels embedded in a gelatinous tunic. The underlying mechanisms regulating programmed cell death and programmed cell removal in this organism are unknown. In this study, we extirpated buds or zooids from B. schlosseri colonies in order to investigate the interplay that exists between buds, zooids, and the vascular system during takeover. Our findings indicate that, in the complete absence of buds (budectomy), organs from adult zooids underwent programmed cell death but were markedly impaired in their ability to be resorbed despite engulfment of zooid-derived cell corpses by phagocytes. However, when buds were removed from only half of the flower-shaped systems of zooids in a colony (hemibudectomy), the budectomized zooids were completely resorbed within 36-48 h following onset of programmed cell death. Furthermore, if hemibudectomies were carried out by using small colonies, leaving only a single functional bud, zooids from the old generation were also resorbed, albeit delayed to 48-60 h following onset of programmed cell death. This bud eventually reached functional maturity, but grew significantly larger in size than any control zooid, and exhibited hyperplasia. This finding strongly suggested that components of the dying zooid viscera could be reutilized by the developing buds, possibly as part of a colony-wide recycling mechanism. In order to test this hypothesis, zooids were surgically removed (zooidectomy) at the onset of takeover, and bud growth was quantitatively determined. In these zooidectomized colonies, bud growth was severely curtailed. In most solitary, long-lived animals, organs and tissues are maintained by processes of continual death and removal of aging cells counterbalanced by regeneration with stem and progenitor cells. In the colonial tunicate B. schlosseri, the same kinds of processes ensure the longevity of the colony (an animal) by cycles of death and regeneration of its constituent zooids (also animals).

Abstract

Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages. CD133(-), CD34(-), and CD45(-) sorted cells (approximately 95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (beta-tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133(+)-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP(+) cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP(+) cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model.

Prospective isolation of human clonogenic common myeloid progenitorsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAManz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L.2002; 99 (18): 11872-11877

Abstract

The hierarchical development from hematopoietic stem cells to mature cells of the hematolymphoid system involves progressive loss of self-renewal capacity, proliferation ability, and lineage potentials. Here we show the prospective isolation of early developmental intermediates, the human clonogenic common myeloid progenitors and their downstream progeny, the granulocyte/macrophage and megakaryocyte/erythrocyte progenitors. All three populations reside in the lineage-negative (lin(-)) CD34(+)CD38(+) fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3R alpha chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling. Multipotent progenitors, early lymphoid progenitors, and the here-defined myeloid progenitors express distinct profiles of hematopoiesis-affiliated genes. The isolation of highly purified hematopoietic intermediates provides tools to better understand developmental programs underlying normal and leukemic hematopoiesis.

Abstract

For me the search for hematopoietic stem cells (HSC) actually started with the discovery by Till, McCulloch, and colleagues (1-3) that bone marrow contained single cells that could give rise to myeloerythroid colonies in the spleen, and sometimes these colonies contained cells that made more spleen colonies as well as radioprotected and reconstituted lethally irradiated mice (3). But in retrospect, it should have started with the remarkable observation of Ray Owen in 1945 that bovine fraternal twins sharing a single placenta and blood circulation retained production of blood cells genetically defined to be from both throughout their life (4). It could be argued that this was the experiment that began both modern experimental hematology as well as modern cellular immunology. The Till, McCulloch, Wu, Becker, and Simonovitch experiments were elegant demonstrations that single, genetically marked cells existed (random DNA breaks and translocations induced by sublethal irradiation of the donor bone marrow) that could both self-renew and differentiate (2, 5). But these experiments did not put the pure cells in the hands of scientists, and so most of their functions for the next 25 years were implied rather than directly analyzed. Just as genetics is the complement to biochemistry (when one considers genes and gene products), cell marking is the complement to cell purification in the fields of developmental and cellular biology. The first attempts at such cellular purification came from the 'school' of Till & McCulloch (6, 7), and independently the school of Van Bekkum in the Netherlands (8). But what was lacking in those experiments and at that time were both a comprehensive approach that would take into account the clonal activity of stem cells in both self-renewal and differentiation to all blood cell outcomes, and the tools with which one could separate what turned out to be an extremely rare population in the bone marrow. And, it wasn't known until much later that most day 8-10 spleen colonies were the progeny of progenitors, not stem cells (9). Two inventions facilitated the technology of purification of HSC: the advent of monoclonal antibody technology by Kohler & Milstein (10), and the development of the multiparameter fluorescence activated cell sorter by the Herzenberg group (11). My laboratory had established assays for the clonal precursors of T cells and B cells, and we had been using the Till-McCulloch spleen colony clonal assays since the mid-1960s. In the late 1970s and early 1980s we began in earnest the search for mouse early hematopoietic progenitors, including HSCs (12-16). The purification of HSCs proved to be much like the purification of an enzyme, or a cell surface receptor, or a gene. Successive enrichments finally led to the isolation of a population, which could no longer be subdivided and which contained precursors that read out in all clonal assays as well as in radioprotection of lethally irradiated hosts (17). Our first experiments transplanting single HSC in 1991 and 1992, led to the definitive demonstration that these were indeed HSCs (18-20). But these experiments and the ideas that led to them were developed in the context of immunology and experimental hematology as they were emerging in the 1950s and 60 s. This volume of Immunological Reviews is a rich testimony to the kinds of ideas and experiments that, at least in retrospect, turned out to be critical. Many roads were taken, but only one ended up at stem cells.

Abstract

Whole-body irradiation at the minimal lethal dose causes bone marrow failure and death within 12-18 days. To identify the principal components of the hematopoietic system that are radioprotective, we transplanted lethally irradiated mice with purified progenitors: common myeloid progenitors (CMPs), megakaryocyte/erythrocyte-restricted progenitors (MEPs), or granulocyte/monocyte-restricted progenitors (GMPs). Transplanted CMPs gave rise to cells both of the granulocyte/monocyte (GM) series and the megakaryocyte/erythrocyte series, whereas GMPs or MEPs showed reconstitution of only GM or ME cells, respectively. CMPs and MEPs but not GMPs protected mice in a dose-dependent manner, suggesting that erythrocytes, platelets, or both are the critical effectors of radioprotection. Accordingly, CMPs and MEPs formed robust colonies in recipient bone marrow and spleen, whereas GMPs formed small colonies that rapidly disappeared. Direct comparisons of spleen CFU (CFU-S) potentials among each progenitor subset showed that MEPs contain the vast majority of day 8 CFU-S activity, suggesting that day 8 CFU-S are the precursors of radioprotective cell subsets. All animals radioprotected for 30 days subsequently survived for at least 6 months post-transplant, and showed only host-derived hematopoiesis after 30 days. These findings suggest that rare hematopoietic stem cells survive myeloablation that can eventually repopulate irradiated hosts if myeloerythroid-restricted progenitors transiently rescue ablated animals through the critical window of bone marrow failure.

Abstract

Hematopoietic stem cells (HSC) have a finite proliferative lifespan, based upon the limited number of times they can be serially transplanted in mice. Telomeres have been shown to shorten during the division of many normal somatic cells in humans, and the attrition of telomeres has been shown to ultimately cause replicative senescence in vitro for a number of different human cell strains. Whereas most human cell types have little to no detectable levels of telomerase activity, hematopoietic cells, including HSC, express low to moderate levels of telomerase, and yet telomeres shorten considerably during replicative aging of these cells. Here we consider the role telomerase may play in the hematopoietic system as well as the effect that over-expression of telomerase reverse transcriptase may have on the replicative capacity of hematopoietic stem cells during transplantation.

Abstract

Although hematopoietic stem cell (HSC) migration into and out of sites of active hematopoiesis is poorly understood, it is a critical process that underlies modern clinical stem cell transplantation and may be important for normal hematopoietic homeostasis. Given the established roles of chemotactic cytokine (chemokine)-directed migration of other leukocyte subsets, the migration of murine HSC to a large panel of CC and CXC chemokines was investigated. HSC migrated only in response to stromal derived factor-1alpha, the ligand for the CXC chemokine receptor 4 (CXCR4). CXCR4 expression by HSC was confirmed by reverse transcription polymerase chain reaction analysis. Surprisingly, HSC also expressed mRNA for CCR3 and CCR9, although they failed to migrate to the ligands for these receptors. The sharply restricted chemotactic responsiveness of HSC is unique among leukocytes and may be necessary for the specific homing of circulating HSC to bone marrow, as well as for the maintenance of HSC in hematopoietic microenvironments.

Abstract

In the adult, tissue-specific stem cells are thought to be responsible for the replacement of differentiated cells within continuously regenerating tissues, such as the liver, skin, and blood system. In this review, we will consider the factors that influence stem cell fate, taking as a primary example the cell fate determination of hematopoietic stem cells.

Abstract

The most immature lymphoid-committed progenitors in both the bone marrow (common lymphoid progenitor) and thymus (proT1) maintain a latent granulocyte/macrophage (G/M) differentiation potential that can be initiated by signals emanating from exogenously expressed IL-2 receptors. In this study, we investigate at which developmental stage thymocytes lose this G/M differentiation potential. We demonstrate that the next maturational stage after proT1 cells (proT2), but not preT (TN3) cells, can convert cell fate from lymphoid to myeloid in response to ectopic IL-2 receptor signaling in human IL-2Rbeta transgenic mice. It is significant that approximately 10% of clonogenic G/M colonies derived from proT cells of IL-2Rbeta transgenic mice have DJ rearrangement specifically at the Dbeta1 but not Dbeta2 segment in the TCRbeta locus. No TCR gene rearrangement is observed in G/M cells from nontransgenic mice, suggesting that the G/M cells we observe in this system were truly lymphoid-committed before stimulation with IL-2. In addition, Dbeta1 and Dbeta2 DJ rearrangement of the TCRbeta gene may be differentially regulated and thus serve as markers for distinct proT cell maturational stages.

Abstract

Although migration of hematopoietic stem cells (HSC) is essential for normal hematopoiesis and successful hematopoietic cell transplantation, little is known about the mechanisms that underlie this movement. We have sought to characterize the factors that regulate HSC migration by analyzing changes in expression of particular adhesion receptors associated with cyclophosphamide/granulocyte colony-stimulating factor (Cy/G-CSF)-induced HSC mobilization.Expression by Lineage(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) HSC of members of the beta1 integrin family of adhesion molecules was assessed in untreated or Cy/G-CSF-treated mice by multiparameter flow cytometry. In parallel, the in vivo homing properties of normal and mobilized HSC were compared following intravenous transfer of fluorescently marked HSC.Normal adult HSC express high levels of several beta1 integrin family members. Following Cy/G treatment, bone marrow HSC selectively downregulate alpha 2 integrin expression and upregulate alpha 5 expression. HSC found in the blood following Cy/G-CSF treatment express significantly lower levels of multiple integrins than their bone marrow and/or splenic counterparts. Changes in integrin expression by blood-borne HSC correlate with a 50% decrease in their ability to home to the bone marrow in short-term assays, and with previously observed defects in competitive engraftment by these HSC. Similar reductions in bone marrow (BM) homing are observed for BM HSC treated with alpha 4 integrin function blocking mAb prior to injection. Modulation of integrin expression induced by mobilization was not associated with cell-cycle progression.Changes in integrin expression and function are associated with HSC mobilization and likely significantly affect the engraftment potential of hematopoietic stem cells.

Abstract

The regulation of hematopoietic stem cell (HSC) homeostasis is not well understood. We screened for genetic polymorphisms that were linked to differences between mouse strains in the numbers of long-term reconstituting HSCs or restricted progenitors in the bone marrow. AKR/J mice had significantly higher frequencies and numbers of both HSCs and restricted progenitors in their bone marrow than C57BL/Ka-Thy-1.1 mice. The C57BL/Ka-Thy-1.1 alleles were partially dominant. A locus on chromosome 17, including the H-2 complex, was significantly linked to the frequency of long-term self-renewing HSCs but showed no evidence of linkage to the frequency of restricted progenitors. Conversely, a chromosome 1 locus exhibited suggestive linkage to restricted progenitor frequencies but was not linked to HSC frequency. This demonstrates that there are distinct genetic determinants of the frequencies of HSCs and restricted progenitors in vivo. The AKR/J chromosome 17 locus was not sufficient to increase HSC frequencies when bred onto a C57BL background. This suggests that to affect HSC frequencies, the product(s) of this locus likely depend on interactions with unlinked modifying loci.

Abstract

Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

Abstract

Within our field, improvement in fluorescence-activated cell sorting (FACS) and molecular technologies has led to various types of correlative studies that imply the developmental sequence and subsequent emigration of thymic-lymphocyte subsets. Unfortunately, the implied conclusions are often accepted unequivocally by most of the immunology community. In fact, direct demonstration of precursor progeny relationships by specific cell marking within the thymus, or specific delivery of purified cells at a particular stage of isolation back into the thymus, are the only methods that reproducibly identify cell stages and intermediates (1-11).

Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChristensen, J. L., Weissman, I. L.2001; 98 (25): 14541-14546

Abstract

Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit(+) (K) lineage(-/lo) (L) Sca-1(+) (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1(lo). c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2(-) HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2(+) multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy(lo) Flk-2(-) (LT-HSCs), Thy(lo) Flk-2(+) (ST-HSCs), and Thy(-) Flk-2(+) multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit(+) lin(-) Sca-1(+) Flk-2(-) from many strains of mice. Fetal liver HSCs are contained within Flk-2(-) and Flk-2(+) KTLS cells.

Abstract

Hematopoietic stem cells (HSCs) reside predominantly in bone marrow, but low numbers of HSCs are also found in peripheral blood. We examined the fate of blood-borne HSCs using genetically marked parabiotic mice, which are surgically conjoined and share a common circulation. Parabionts rapidly established stable, functional cross engraftment of partner-derived HSCs and maintained partner-derived hematopoiesis after surgical separation. Determination of the residence time of injected blood-borne progenitor cells suggests that circulating HSCs/progenitors are cleared quickly from the blood. These data demonstrate that HSCs rapidly and constitutively migrate through the blood and play a physiological role in, at least, the functional reengraftment of unconditioned bone marrow.

Abstract

Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.

Abstract

The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition.

Abstract

The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.

Abstract

Hematopoietic cell transplantation has progressed from the use of unpurified bone marrow cells or mobilized peripheral blood cells to the use of purified stem cells and progenitor cells. These kinds of transplants can be designed to provide not only hematopoietic rescue but also augmented innate and acquired immunity.

Abstract

Hematopoietic fate maps in the developing mouse embryo remain imprecise. Definitive, adult-type hematopoiesis first appears in the fetal liver, then progresses to the spleen and bone marrow. Clonogenic common lymphoid progenitors and clonogenic common myeloid progenitors (CMPs) in adult mouse bone marrow that give rise to all lymphoid and myeloid lineages, respectively, have recently been identified. Here it is shown that myelopoiesis in the fetal liver similarly proceeds through a CMP equivalent. Fetal liver CMPs give rise to megakaryocyte-erythrocyte-restricted progenitors (MEPs) and granulocyte-monocyte-restricted progenitors (GMPs) that can also be prospectively isolated by cell surface phenotype. MEPs and GMPs generate mutually exclusive cell types in clonogenic colony assays and in transplantation experiments, suggesting that the lineage restriction observed within each progenitor subset is absolute under normal conditions. Purified progenitor populations were used to analyze expression profiles of various hematopoiesis-related genes. Expression patterns closely matched those of the adult counterpart populations. These results suggest that adult hematopoietic hierarchies are determined early in the development of the definitive immune system and suggest that the molecular mechanisms underlying cell fate decisions within the myeloerythroid lineages are conserved from embryo to adult. (Blood. 2001;98:627-635)

Abstract

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present in Pbx1(-/-) fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1(-/-) embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted in Pbx1(-/-) embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated. (Blood. 2001;98:618-626)

Abstract

It is reasonable to propose that gene expression profiles of purified stem cells could give clues for the molecular mechanisms of stem cell behavior. We took advantage of cDNA subtraction to identify a set of genes selectively expressed in mouse adult hematopoietic stem cells (HSC) as opposed to bone marrow (BM). Analysis of HSC-enriched genes revealed several key regulatory gene candidates, including two novel seven transmembrane (7TM) receptors. Furthermore, by using cDNA microarray techniques we found a large set of HSC-enriched genes that are expressed in mouse neurospheres (a population greatly enriched for neural progenitor cells), but not present in terminally differentiated neural cells. In situ hybridization demonstrated that many of them, including one HSC-enriched 7TM receptor, were selectively expressed in the germinal zones of fetal and adult brain, the regions harboring mouse neural stem cells. We propose that at least some of the transcripts that are selectively and commonly expressed in two or more types of stem cells define a functionally conserved group of genes evolved to participate in basic stem cell functions, including stem cell self-renewal.

Abstract

It has been proposed that there are at least 2 classes of dendritic cells (DCs), CD8alpha(+) DCs derived from the lymphoid lineage and CD8alpha(-) DCs derived from the myeloid lineage. Here, the abilities of lymphoid- and myeloid-restricted progenitors to generate DCs are compared, and their overall contributions to the DC compartment are evaluated. It has previously been shown that primitive myeloid-committed progenitors (common myeloid progenitors [CMPs]) are efficient precursors of both CD8alpha(+) and CD8alpha(-) DCs in vivo. Here it is shown that the earliest lymphoid-committed progenitors (common lymphoid progenitors [CLPs]) and CMPs and their progeny granulocyte-macrophage progenitors (GMPs) can give rise to functional DCs in vitro and in vivo. CLPs are more efficient in generating DCs than their T-lineage descendants, the early thymocyte progenitors and pro-T cells, and CMPs are more efficient DC precursors than the descendant GMPs, whereas pro-B cells and megakaryocyte-erythrocyte progenitors are incapable of generating DCs. Thus, DC developmental potential is preserved during T- but not B-lymphoid differentiation from CLP and during granulocyte-macrophage but not megakaryocyte-erythrocyte development from CMP. In vivo reconstitution experiments show that CLPs and CMPs can reconstitute CD8alpha(+) and CD8alpha(-) DCs with similar efficiency on a per cell basis. However, CMPs are 10-fold more numerous than CLPs, suggesting that at steady state, CLPs provide only a minority of splenic DCs and approximately half the DCs in thymus, whereas most DCs, including CD8alpha(+) and CD8alpha(-) subtypes, are of myeloid origin. (Blood. 2001;97:3333-3341)

Abstract

We identified an IL-7Ralpha(+)Sca-1(low)c-Kit(low) population in E14 fetal liver, which is the phenotypical analog of common lymphoid progenitors (CLP) in adult bone marrow. After transfer into newborn mice, the IL-7Ralpha(+)Sca-1(low)c-Kit(low) population rapidly differentiated into CD45(+)CD4(+)CD3(-) cells, which are candidate cells for initiating lymph node and Peyer's patch formation. In addition, this population also gave rise to B, T, NK, and CD8alpha(+) and CD8alpha(-) dendritic cells. The fetal liver precursors expressed a significantly lower level of the myeloid-suppressing transcription factor Pax-5, than adult CLP, and retained differentiation activity for macrophages in vitro. We propose that the transition from fetal liver IL-7Ralpha(+)Sca-1(low)c-Kit(low) cells to adult CLP involves a regulated restriction of their developmental potential, controlled, at least in part, by Pax-5 expression.

Abstract

Reactivation of telomerase and maintenance of telomere length can lead to the prevention of replicative senescence in some human somatic cells grown in vitro. To investigate whether telomere shortening might also play a role in the limitation of hematopoietic stem cell (HSC) division capacity in vivo, we analyzed telomere length during serial transplantation of murine HSCs. Southern blot analysis of telomere length in donor bone marrow cells revealed extensive shortening ( approximately 7 kb) after just two rounds of HSC transplantation. The number of cycling HSCs increased after transplantation and remained elevated for at least 4 mo, while the frequency of HSCs in the bone marrow was completely regenerated by 2 mo after transplantation. Direct analysis of telomeres in HSCs by fluorescent in situ hybridization during serial transplantation also revealed a reduction in telomere size. Together, these data show that telomeres shorten during division of HSCs in vivo, and are consistent with the hypothesis that telomere shortening may limit the replicative capacity of HSCs.

Abstract

Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB HSC) are widely used for transplantation in the treatment of malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G(0) or G(1) phase of the cell cycle. This has raised the question of whether a subset of noncycling BM HSC is selectively released, or whether cycling BM HSC are mobilized after M phase, but before the next S phase of the cell cycle. To distinguish between these possibilities, mice were treated with one dose of CY followed by daily doses of G-CSF, and dividing cells were marked by administration of bromodeoxyuridine (BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB (LT)-HSC stained positively for BrdU, and therefore derived from cells that divided during the treatment interval. Next, LT-HSC from mice previously treated with a single dose of CY, which kills cycling cells, and 3 daily doses of G-CSF, were nearly all killed by a second dose of CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle. Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA content of MPB HSC suggests that these cells are mostly in G(1) phase. After CY/G-CSF treatment, virtually all BM LT-HSC enter the cell cycle; some of these HSC then migrate into the blood, specifically after M phase, and are rapidly recruited to particular hematopoietic organs.

L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesisPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAQian, F., Hanahan, D., Weissman, I. L.2001; 98 (7): 3976-3981

Abstract

L-selectin mediates homing of lymphocytes to lymph nodes (LN). Transgenic mice that express rat insulin promoter regulated simian virus 40 Tag (RIP-Tag) develop large, local cancers that metastasize to liver but not LN. To test whether this lack of LN metastases reflects their absence from the circulation, transgenic mice were produced that express Tag (T), L-selectin (L), and Escherichia coli LacZ (Z), in pancreatic beta cells. LTZ mice developed insulinomas that specifically had LN metastases; metastasis was blocked by an anti L-selectin mAb. LacZ(+) tumor cells from these LN homed to secondary LN upon transfer. These results suggest that the highly vascularized islet carcinomas are shedding tumor cells into the bloodstream, which is a necessary but insufficient condition for metastasis to occur; L-selectin can facilitate homing of such tumor cells to LN, resulting in metastasis.

Abstract

The promyelocytic leukemia retinoic acid receptor alpha (PMLRARalpha) chimeric protein is associated with acute promyelocytic leukemia (APL). PMLRARalpha transgenic mice develop leukemia only after several months, suggesting that PMLRARalpha does not by itself confer a fully malignant phenotype. Suppression of apoptosis can have a central role in tumorigenesis; therefore, we assessed whether BCL-2 influenced the ability of PMLRARalpha to initiate leukemia. Evaluation of preleukemic animals showed that whereas PMLRARalpha alone modestly altered neutrophil maturation, the combination of PMLRARalpha and BCL-2 caused a marked accumulation of immature myeloid cells in bone marrow. Leukemias developed more rapidly in mice coexpressing PMLRARalpha and BCL-2 than in mice expressing PMLRARalpha alone, and all mice expressing both transgenes succumbed to leukemia by 7 mo. Although both preleukemic, doubly transgenic mice and leukemic animals had abundant promyelocytes in the bone marrow, only leukemic mice exhibited thrombocytopenia and dissemination of immature cells. Recurrent gain of chromosomes 7, 8, 10, and 15 and recurrent loss of chromosome 2 were identified in the leukemias. These chromosomal changes may be responsible for the suppression of normal hematopoiesis and dissemination characteristic of the acute leukemias. Our results indicate that genetic changes that inhibit apoptosis can cooperate with PMLRARalpha to initiate APL.

Abstract

Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.

Abstract

A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH(2) and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and short-term hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the alpha subunit of both G(i) and G(q) subfamilies. The results of in vitro GTPase single-turnover assays using Galpha(i) indicated that RGS18 accelerates the intrinsic GTPase activity of Galpha(i). Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on G(q)-mediated signaling pathways in vivo.

Abstract

Multipotent stem cells are clonal cells that self-renew as well as differentiate to regenerate adult tissues. Whereas stem cells and their fates are known by unique genetic marker studies, the fate and function of these cells are best studied by their prospective isolation. This review is about the properties of various highly purified tissue-specific multipotent stem cells and purified oligolineage progenitors. We contend that unless the stem or progenitor cells in question have been purified to near homogeneity, one cannot know whether their generation of expected (or unexpected) progeny is a property of a known cell type. It is interesting that in the hematopoietic system the only long-term self-renewing cells in the stem and progenitors pool are the hematopoietic stem cells. This fact is discussed in the context of normal and leukemic hematopoiesis.

Abstract

Dendritic cells (DCs) are professional antigen-presenting cells which both initiate adaptive immune responses and control tolerance to self-antigens. It has been suggested that these different effects on responder cells depend on subsets of DCs arising from either myeloid or lymphoid hematopoietic origins. In this model, CD8 alpha+ Mac-1- DCs are supposed to be of lymphoid while CD8 alpha- Mac-1+ DCs are supposed to be of myeloid origin. Here we summarize our findings that both CD8 alpha+ and CD8 alpha- DCs can arise from clonogenic common myeloid progenitors (CMPs) in both thymus and spleen. Therefore CD8 alpha expression DCs does not indicate a lymphoid origin and differences among CD8 alpha+ and CD8 alpha- DCs might rather reflect maturation status than ontogeny. On the basis of transplantation studies, it seems likely that most of the DCs in secondary lymphoid organs and a substantial fraction of thymic DCs are myeloid-derived.

Abstract

Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133)(+), 5E12(+), CD34(-), CD45(-), and CD24(-/lo). Single CD133(+) CD34(-) CD45(-) sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133(+) CD34(-) CD45(-) cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.

Abstract

Growth factors can cause cells to proliferate, differentiate, survive, or die. Distinguishing between these responses is difficult in multicellular, multiparameter systems. Yet this is essential to understand the impact on cells like hematopoietic stem cells (HSCs), which have strict and still poorly understood growth factor requirements. Single cell plating in serum-free medium allows direct assessment of growth factor responses. The range of tested factors can be expanded if the cells are protected from growth factor deprivation-induced apoptosis. BCL-2 is overexpressed in HSCs of H2K-BCL-2 transgenic mice, protecting them from many apoptotic stimuli. The response of single wild-type and transgenic HSCs to stimulations with individual factors was tested. Surprisingly, we find that high level BCL-2 expression does not prevent rapid death under serum-free conditions, even though it does in the presence of serum. We also find that transgenic, but not wild-type cells, survive and proliferate rapidly in response to steel factor (Kit ligand). These studies show that two separate signals are necessary to prevent apoptosis in HSCs, and that Kit ligand by itself provides a strong proliferative stimulus to HSCs. However, the proliferative response does not result in self-renewal, but in differentiation to all known hematopoietic oligolineage progenitors.

Abstract

Dendritic cells (DCs) are critical in both initiating adaptive immune responses and maintaining tolerance to self antigens. These apparently contradictory roles have been suggested to depend on different subsets of DCs that arise from either myeloid or lymphoid hematopoietic origins, respectively. Although DC expression of CD8alpha is attributed to a lymphoid origin, here we show that both CD8alpha+ and CD8alpha- DCs can arise from clonogenic common myeloid progenitors in both thymus and spleen. Thus, expression of CD8alpha is not indicative of a lymphoid origin, and phenotypic and functional differences among DC subsets are likely to reflect maturation status rather than ontogeny.

Abstract

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

Abstract

The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.

Abstract

The primary role of cytokines in haemato-lymphopoiesis is thought to be the regulation of cell growth and survival. But the instructive action of cytokines in haematopoiesis has not been well addressed. Here we show that a clonogenic common lymphoid progenitor, a bone marrow-resident cell that gives rise exclusively to lymphocytes (T, B and natural killer cells), can be redirected to the myeloid lineage by stimulation through exogenously expressed interleukin (IL)-2 and GM-CSF (granulocyte/macrophage colony-stimulating factor) receptors. Analysis of mutants of the beta-chain of the IL-2 receptor revealed that the granulocyte- and monocyte-differentiation signals are triggered by different cytoplasmic domains, showing that the signalling pathway(s) responsible for these unique developmental outcomes are separable. Finally, we show that the endogenous myelomonocytic cytokine receptors for GM-CSF and macrophage colony-stimulating factor (M-CSF) are expressed at low to moderate levels on the more primitive haematopoietic stem cells, are absent on common lymphoid progenitors, and are upregulated after myeloid lineage induction by IL-2. We conclude that cytokine signalling can regulate cell-fate decisions and propose that a critical step in lymphoid commitment is downregulation of cytokine receptors that drive myeloid cell development.

Abstract

Engraftment of allogeneic bone marrow (BM) has been shown to induce tolerance to organs genotypically matched with the BM donor. Immune reconstitution after BM transplantation therefore involves re-establishment of a T cell pool tolerant to antigens present on both donor and host tissues. However, how hematopoietic grafts exert their influence over the regenerating immune system is not completely understood. Prior studies suggest that education of the newly arising T cell pool involves distinct contributions from donor and host stromal elements. Specifically, negative selection is thought to be mediated primarily by donor BM-derived antigen-presenting cells, whereas positive selection is dictated by radio-resistant host-derived thymic stromal cells. In this report we studied the effect of highly purified allogeneic hematopoietic stem cells (HSCs) on organ transplantation tolerance induction and immune reconstitution. In contrast to engraftment of BM that results in near-complete donor T cell chimerism, HSC engraftment results in mixed T cell chimerism. Nonetheless we observed that HSC grafts induce tolerance to donor-matched neonatal heart grafts, and one way the HSC grafts alter host immune responses is via deletion of newly arising donor as well as radiation-resistant host T cells. Furthermore, using an in vivo assay of graft rejection to study positive selection we made the unexpected observation that T cells in chimeric mice rejected grafts only in the context of the donor MHC type. These latter findings conflict with the conventionally held view that radio-resistant host elements primarily dictate positive selection.

Abstract

Hematopoietic stem cell gene therapy holds promise for the treatment of many hematologic disorders. One major variable that has limited the overall success of gene therapy to date is the lack of sustained gene expression from viral vectors in transduced stem cell populations. To understand the basis for reduced gene expression at a single-cell level, we have used a murine retroviral vector, MFG, that expresses the green fluorescent protein (GFP) to transduce purified populations of long-term self-renewing hematopoietic stem cells (LT-HSC) isolated using the fluorescence-activated cell sorter. Limiting dilution reconstitution of lethally irradiated recipient mice with 100% transduced, GFP(+) LT-HSC showed that silencing of gene expression occurred rapidly in most integration events at the LT-HSC level, irrespective of the initial levels of GFP expression. When inactivation occurred at the LT-HSC level, there was no GFP expression in any hematopoietic lineage clonally derived from silenced LT-HSC. Inactivation downstream of LT-HSC that stably expressed GFP( )in long-term reconstituted animals was restricted primarily to lymphoid cells. These observations suggest at least 2 distinct mechanisms of silencing retrovirally expressed genes in hematopoietic cells.

AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocationPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMiyamoto, T., Weissman, I. L., Akashi, K.2000; 97 (13): 7521-7526

Abstract

Leukemia-specific AML1/ETO transcripts are detectable in most patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. To understand the inconsistency between the clinical cure and the presence of "residual disease" at a molecular level, we separated and identified the cells expressing AML1/ETO by phenotype and function. Here we demonstrate that AML1/ETO transcripts are present in a fraction of stem cells, monocytes, and B cells in remission marrow, and in a fraction of B cells in leukemic marrow, but not in T cells. AML1/ETO transcripts also were demonstrated in a fraction of colony-forming cells of erythroid, granulocyte-macrophage, and/or megakaryocyte lineages in both leukemic and remission marrow. These data strongly suggest that the acquisition of the t(8;21) occurs at the level of stem cells capable of differentiating into B cells as well as all myeloid lineages, and that a fraction of the AML1/ETO-expressing stem cells undergo additional oncogenic event(s) that ultimately leads to transformation into AML.

50 million years of chordate evolution: Seeking the origins of adaptive immunityPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICALaird, D. J., De Tomaso, A. W., COOPER, M. D., Weissman, I. L.2000; 97 (13): 6924-6926

Abstract

Botryllus schlosseri is a clonally modular ascidian, in which individuals (zooids) have a finite life span that is intimately associated with a weekly budding process called blastogenesis. Every blastogenic cycle concludes with a synchronized phase of regression called takeover, during which all zooids in a colony die, primarily by apoptosis, and are replaced by a new generation of asexually derived zooids. We have previously documented that, in addition to this cyclical death phase, entire colonies undergo senescence during which all asexually derived individuals in a colony, buds and zooids, die in concert. In addition, when a specific parent colony (genet) is experimentally separated into a number of clonal replicates (ramets), ramets frequently undergo senescence simultaneously, indicating that mortality can manifest itself in nonrandom fashion. Here, we document a morphological portrait of senescence in laboratory-maintained colonies from Monterey Bay, California, that exhibit nonrandom mortality. Nonrandom senescence proceeded according to a series of characteristic changes within the colony over a period of about one week. These changes included systemic constriction and congestion of the vasculature accompanied by massive accumulation of pigment cells in the zooid body wall (mantle), blood vessels, and ampullae; gradual shrinkage of individual zooids; loss of colonial architecture, and ultimately death. At the ultrastructural level, individual cells exhibited changes typical of ischemic cell death, culminating in necrotic cell lysis rather than apoptosis. Collectively, these observations indicate that senescence is accompanied by unique morphological changes that occur systemically, and which are distinct from those occurring during takeover. We discuss our findings in relation to current experimental models of aging and the possible role of a humoral factor in bringing about the onset of senescence.

Abstract

The thymus has been regarded as the major site of T cell differentiation. We find that in addition to alphabeta and gammadelta T cells, a significant number (approximately 3 x 104 per day) of B220+IgM+ mature B cells are exported from the thymus of C57BL/6 mice. Of these emigrating B cells, we estimate that at least approximately 2 x 104 per day are cells which developed intrathymically, whereas a maximum of approximately 0.8 x 104 per day are cells which circulated through the thymus from the periphery. The thymus possesses a significant number of pro-B and pre-B cells that express CD19, VpreB, lambda5, and pax-5. These B cell progenitors were found in the thymic cortex, whereas increasingly mature B cells were found in the corticomedullar and medullary regions. Other lymphoid cells, including NK cells and lymphoid dendritic cells, are not exported from the thymus at detectable levels. Thus, the thymus contributes to the formation of peripheral pools of B cells as well as of alphabeta and gammadelta T cells.

Abstract

The antigen specificity of a rat monoclonal antibody TER-119 was investigated. In adult mice, TER-119 reacted with mature erythrocytes, 20-25% of bone marrow cells and 2-3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30-40% of d 10 yolk sac cells, 80-90% of d 14 fetal liver cells and 40-50% of newborn liver cells were reactive with TER-119. TER-119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac-1, Gr-1) or B-cell (B220) markers. Morphological examination and haematopoietic colony-forming assays for isolated TER-119+ cells revealed that TER-119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) activities. Erythroleukaemia cell lines do not express the TER-119 antigen even after stimulation with dimethylsulphoxide. TER-119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52-kDa band was detected by TER-119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER-119 antigen is a molecule associated with cell-surface glycophorin A but not with glycophorin A itself.

Abstract

Lymphopoiesis of mature and diverse populations of T, B and NK (natural killer) cells from multipotent hematopoietic stem cells is an ideal model of tissue generation and regeneration. Identification and isolation of hematolymphoid stem and progenitor cells in several laboratories over the past several years have provided populations that can be studied biologically for lineage commitment and biochemically for receptor function, signal transduction and selective gene expression. These studies may ultimately provide candidate genes involved in lineage commitment, cell death or survival, self-renewal and migratory capacities of progenitors.

Abstract

Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1(+) CD3(-) cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1(+) CD3(-) cells were responsible for acute tumor rejection, establishing the relationship of NK1.1(+) CD3(-) cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNgamma in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.

Abstract

Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.

Abstract

Stem cells are the natural units of embryonic generation, and also adult regeneration, of a variety of tissues. Recently, the list of tissues that use the model of differentiation from stem to progenitor to mature cell has increased from blood to include a variety of tissues, including both central and peripheral nervous systems and skeletal muscle; it is also possible that all organs and tissues are derived from, and still contain, stem cells. Because the number and activities of stem cells and their progeny are homeostatically regulated, clinical stem cell transplantation could greatly add to the physician's armamentarium against degenerative diseases.

The role of apoptosis in the regulation of hematopoietic stem cells: Overexpression of BCL-2 increases both their number and repopulation potentialJOURNAL OF EXPERIMENTAL MEDICINEDomen, J., Cheshier, S. H., Weissman, I. L.2000; 191 (2): 253-263

Abstract

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2-transgenic mice have increased numbers of HSC in bone marrow (2.4x wild type), but fewer of these cells are in the S/G(2)/M phases of the cell cycle (0.6x wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

Abstract

We report here the transplantation of extensively purified "mobilized" peripheral blood CD34Thy-1 hematopoietic stem cells from 22 patients with recurrent or metastatic breast cancer. Patients were mobilized with either high-dose granulocyte colony-stimulating factor (G-CSF) alone or cyclophosphamide plus G-CSE Median purity of the stem cell product at cryopreservation was 95.3% (range, 91.1%-98.3%), and viability was 98.6% (range, 96.5%-100%). After high-dose chemotherapy with carmustine, cisplatin, and cyclophosphamide, CD34+Thy-1 cells at a median dose of 11.3 x 10(5) per kilogram (range, 4.7-163 x 10(5) per kilogram) were infused. No infusion-related toxicity was observed. Neutrophil recovery was prompt, with median absolute neutrophil count >500/microL by day 10 (range, 8-15 days) and >1000/microL by day 11 (range, 8-17 days). Median platelet recovery (>20,000/microL) was observed by day 14 (range, 9-42 days) and >50,000/microL by day 17 (range, 11-49 days). Tumor cell depletion below the limits of detection of a sensitive immunofluorescence-based assay was accomplished in all patients who had detectable tumor cells in apheresis products before processing. Although CD4+ T-cell reconstitution was slow, no unusual infections were observed. Neither early nor late graft failure was observed, and no patient required infusion of unmanipulated backup cells. At a median follow-up of approximately 1.4 years and a maximum follow-up of 2.5 years, 16 of the 22 patients remain alive, with 9 free of disease progression, and have stable blood counts. In summary, highly purified CD34+Thy-1+ cells used as the sole source of the hematopoietic graft result in rapid and sustained hematopoietic engraftment.

Abstract

Although purified hematopoietic stem cells (HSC) are sufficient to engraft irradiated allogeneic recipients, bone marrow (BM) contains other cells that facilitate engraftment. Here, several candidate facilitators were tested by cotransplantation with HSC. Both TCR+ and TCR- CD8alpha+ BM subpopulations have facilitative potential. CD8+TCR+ cells are typical T lymphocytes. CD8+TCR- facilitators are CD3 , not CD3+, have a granular morphology, and are CD8beta- and CD11c+; they share phenotypic characteristics with CD8(alpha)alpha lymphoid dendritic cells and veto cells. We also demonstrate that lytic function is nqt necessary for facilitation and that the CD8alpha molecule is either important for facilitation or in the development of facilitators.

Heritable germ and somatic cell lineage competitions in chimeric colonial protochordatesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAStoner, D. S., Rinkevich, B., Weissman, I. L.1999; 96 (16): 9148-9153

Abstract

Theories of evolution that state natural selection acts on individuals have been modified to include multiple levels of selection. Here we demonstrate in chimeric protochordates that primitive germ cell (pgc) and somatic cell (psc) lineages have traits that also make them likely units of natural selection. Specifically, by using microsatellites to determine the genetic identity of various somatic and gametic tissues within vascularly fused Botryllus schlosseri chimeras, we show that genetically distinct pgc and psc can compete for access to developing gonads and somatic organs, and that this competition is hierarchical, reproducible, and heritable. Given that a single, highly polymorphic locus (Fu/HC) controls whether two contacting colonies fuse or reject, our findings also support a leading hypothesis for why the highly polymorphic histocompatibility loci common to many metazoa may have arisen or been maintained: to limit supercompetitor lineages to histocompatible kin.

Abstract

IL-7 receptor (IL-7R) plays critical roles in lymphocyte development by promoting survival and proliferation and by inducing V(D)J recombination in TCR and Ig loci. Here, we demonstrate that IL-7R-activated Stat5 binds to consensus motifs in the 5' regions of Jgamma segments and induces germline transcripts. We also show that a constitutively active form of Stat5 restores V-J recombination of TCRgamma genes and partially rescues T cell development from IL-7R(-/-) T cell precursors, especially in favor of gammadelta T cells. Therefore, this study reveals a potential role of Stat5 in T cell development and also implies that IL-7R may control the accessibility of the TCRgamma locus through Stat5-induced germline transcription.

Antibodies to CD44 and integrin alpha(4), but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitmentPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABrocke, S., Piercy, C., Steinman, L., Weissman, I. L., VEROMAA, T.1999; 96 (12): 6896-6901

Abstract

The role of various adhesion molecules in lymphocyte homing to the brain and in inflammatory autoimmune disease of the central nervous system (CNS) was examined in mice. Activated T cell lines and clones expressed CD44 and integrin alpha4, but not L-selectin, and entered the CNS independent of their antigen specificity. mAbs directed against CD44 and integrin alpha4 prevented the transfer of experimental autoimmune encephalomyelitis (EAE) by myelin basic protein-specific T cells. T cells preincubated with anti-CD44 or antiintegrin alpha4 were blocked only partially from entering the brain parenchyma. However, both antibodies efficiently prevented CNS inflammation and clinical expression of EAE when injected in vivo. This effect lasted as long as antibodies were administered. Antibodies specific for L-selectin had no effect on homing of encephalitogenic T cells to the brain or development of EAE. Antiintegrin alpha4 and anti-CD44 did not impair the activation and function of encephalitogenic T cells in vitro and did not deplete integrin alpha4- or CD44-positive cells in vivo. These data suggest that, in the absence of leukocyte recruitment, the entry of a reduced number of activated myelin basic protein-reactive T cells in the CNS is not sufficient for the development and expression of EAE. We propose that antibodies to integrin alpha4 and CD44 prevent clinical disease by partially targeting the primary influx of encephalitogenic T cells and by preventing the secondary influx of leukocytes to lesions initiated by the transferred T cells.

Abstract

Mechanisms and pathways for commitment to the lymphoid lineage from hematopoietic stem cells (HSC) remain controversial. The interleukin-7 receptor (IL-7R) transduces nonredundant signals for both T- and B-cell development. Recently, we identified a clonogenic common lymphoid progenitor population in mouse bone marrow that can give rise to T, B, and natural killer (NK) cells, but lacks myeloid differentiation capacity. These cells are not self-renewing stem cells, but progenitors that have a limited life span. HSC do not express IL-7R, and the upregulation of the IL-7R occurs at the stage of common lymphoid progenitors. The IL-7R mediates nonredundant signals to reinforce the survival of developing T cells, and to promote rearrangement of immunoglobulin heavy chain genes in B-cell progenitors. Thus, common lymphoid progenitors exist in early hematopoiesis, and expression of the IL-7R is a critical step in the initiation of lymphoid development from HSC.

Abstract

Hematopoietic stem cells (HSCs) are the rare cells from which all hematopoietic cells are derived. The absence of HSCs is not compatible with life because many essential cells, such as myeloid and erythroid cells, are short lived. The hematopoietic system is the first essential organ system that fails following cytotoxic treatments. It is the vulnerability of HSCs that prevents regeneration following treatment and thus long-term survival. Because HSCs have the capacity to regenerate a functional hematopoietic system, the manipulation of these cells in vitro holds many promises for gene-therapeutic and other applications; however, these are severely curtailed by current difficulties in maintaining and expanding HSCs in culture. This review focuses on recent approaches towards understanding how the HSC compartment is regulated in vivo and discusses how this knowledge might be applied to manipulating HSC numbers.

In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICACheshier, S. P., Morrison, S. J., Liao, X. S., Weissman, I. L.1999; 96 (6): 3120-3125

Abstract

A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, approximately 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1 phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although approximately 75% of LT-HSC are quiescent in G0 at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.

Cyclophilin C-associated protein: A normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivoPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICATrahey, M., Weissman, I. L.1999; 96 (6): 3006-3011

Abstract

Mouse cyclophilin C-associated protein (CyCAP) is a member of the scavenger-receptor cysteine-rich domain superfamily and is 69% identical to the human Mac-2 binding protein. Here, we show that CyCAP is a widely expressed secreted glycoprotein that modulates the host response to endotoxin. Gene-targeted CyCAP-deficient mice are more sensitive to the lethal effects of endotoxin. In response to endotoxin, CyCAP-deficient mice overproduced interleukin 12 and interferon-gamma systemically and tumor necrosis factor alpha locally; these are proinflammatory molecules that also promote T helper 1 responses. Furthermore, macrophages stimulated in vitro with endotoxin in serum deficient in CyCAP secreted more tumor necrosis factor alpha, supporting the proposal that CyCAP specifically down-modulates endotoxin signaling.

Abstract

The MHC molecules have been historically perceived as transplantation antigens, though it is now recognized that their primary, if not sole, role is in eliminating parasites and in surveillance and clearance of aberrant self. Indeed, pregnancy in mammals would represent the closest to a natural transplantation process that occurs in vertebrates. However, among the immediate ancestors to the vertebrates, natural intraspecific allorecognition processes are common. Among members of the colonial tunicate Botryllus schlosseri, two individuals that share a single allele of the highly polymorphic fusibility/histocompatibility (Fu/HC) locus are able to fuse with one another. Could this Fu/HC be related to the MHC such that the MHC really did have its origins as a transplantation antigen? Presently we review the genetics and biology of natural transplantation processes in colonial tunicates, comparing it with allorecognition as mediated through the vertebrate T-cell receptor, killer cell inhibitory receptor/Ly49, and MHC. Experimental approaches to determining if the molecules regulating allorecognition in tunicates have any ancestral relationship to the vertebrate MHC are discussed, as is a genomic approach to isolating novel mediators of allorecognition. We also explore the biological basis for allorecognition in colonial tunicates and recent work that highlights the costs of not maintaining a system for allorecognition.

Abstract

To analyze whether the phenotypic abnormalities observed in lymphotoxin-alpha(-/-) (LT alpha-/-) mice are intrinsic to the hemolymphoid system itself or dependent on stromal elements, wild-type (WT) mice were reconstituted with bone marrow (BM) cells enriched for hemopoietic stem cells from LT alpha-/- animals. WT mice reconstituted with LT alpha-/- c-kit+ Lin- Sca-1+ BM cells do not maintain follicular dendritic cell (FDC) networks and do not form primary follicles, while clear segregation of B and T cells could be observed. Furthermore, IgM+ IgD- B cells, MOMA-1 (anti-metallophilic macrophages), ERTR-9 (anti-marginal zone macrophages), and MECA-367 (anti-MAdCAM-1) were all absent from the splenic marginal zone. Surprisingly, however, the expression of MOMA-1, ERTR-9, and MAdCAM-1 was normal in the lymph nodes of mice reconstituted with LT alpha-/- cells. In addition, peanut agglutinin-positive germinal centers were observed in both the spleen and mesenteric lymph nodes, although in the absence of detectable FDC. Furthermore, in animals reconstituted with a mixture of LT alpha-/- and WT c-kit+ Lin- Sca-1+, GC contained either predominantly LT alpha-/- B cells or WT B cells. These results suggest that although the formation of primary follicles, FDC networks, and the splenic marginal zone are all dependent on hemopoietically derived LT alpha, germinal center formation and the expression of MAdCAM-1, MOMA-1, and ERTR-9 in lymph nodes are not. Our results also suggest that the disturbed B-T cell separation in LT alpha-/- mice is unrelated to defects in the marginal zone.

Abstract

All lymphocytes are derived from hematopoietic stem cells (HSC). The interleukin-7 receptor (IL-7R) transduces non-redundant signals for both T and B-cell development from HSC. The upregulation of the IL-7R occurs at the stage of the clonogenic common lymphoid progenitor, a recently identified population that can give rise to all lymphoid lineages (T, B and natural killer cells) at a single cell level. The IL-7R plays a critical role in the rearrangement of immunoglobulin heavy chain genes required for B-cell development. IL-7R expression is critically regulated in developing thymocytes; thymocytes that fail the positive selection process downregulate the IL-7R, but those undergoing positive selection upregulate or maintain IL-7R expression. Recent data indicate that IL-7 signaling enhances the survival of developing thymocytes and mature T cells, presumably by its upregulating Bcl-2. Detailed analysis of the signaling cascades activated by the IL-7R may help to reveal the differential roles of IL-7 signaling in T and B-cell development.

Abstract

The colonial urochordate Botryllus schlosseri undergoes a genetically defined, natural transplantation reaction that is controlled by a single Mendelian locus (called the Fu/HC). This Fu/HC-based allorecognition system is initiated when peripheral elements of the vasculature interact on the edges of two asexually expanding colonies. To better understand the spatial organization of the cellular elements responsible for Fu/HC-based allorecognition, we bypassed the normal site of interaction (the ampullae) and experimentally transplanted zooids between Fu/HC-noncompatible Botryllus schlosseri pairs. The results show that (1) instead of the expected rejections (tissue necroses) that develop after natural contacts between peripheral blood vessels, the transplanted organs are morphologically eliminated within a few days in conjunction with the normal blastogenic cycle; and (2) donor-recipient chimerism is established after complete morphological elimination of transplanted tissues. These results suggest that Fu/HC-based allorecognition responses in Botryllus schlosseri occur exclusively at the ampullae and that once cells have crossed this barrier, they are able to survive and proliferate in the new host colony.

The PEBP2 beta MYH11 fusion created by Inv(16)(p13;q22) in myeloid leukemia impairs neutrophil maturation and contributes to granulocytic dysplasiaPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKogan, S. C., Lagasse, E., Atwater, S., Bae, S. C., Weissman, I., Ito, Y., BISHOP, J. M.1998; 95 (20): 11863-11868

Abstract

Chromosomal translocations involving the genes encoding the alpha and beta subunits of the Pebp2/Cbf transcription factor have been associated with human acute myeloid leukemia and the preleukemic condition, myelodysplasia. Inv(16)(p13;q22) fuses the gene encoding the beta subunit of Pebp2 to the MYH11 gene encoding a smooth muscle myosin heavy chain (Smmhc). To examine the effect of the inv(16)(p13;q22) on myelopoiesis, we used the hMRP8 promoter element to generate transgenic mice expressing the Pebp2betaSmmhc chimeric fusion protein in myeloid cells. Neutrophil maturation was impaired in PEBP2betaMYH11 transgenic mice. Although the transgenic mice had normal numbers of circulating neutrophils, their bone marrow contained increased numbers of immature neutrophilic cells, which exhibited abnormal characteristics. In addition, PEBP2betaMYH11 inhibited neutrophilic differentiation in colonies derived from hematopoietic progenitors. Coexpression of both PEBP2betaMYH11 and activated NRAS induced a more severe phenotype characterized by abnormal nuclear morphology indicative of granulocytic dysplasia. These results show that PEBP2betaMYH11 can impair neutrophil development and provide evidence that alterations of Pebp2 can contribute to the genesis of myelodysplasia.

Abstract

Recent studies have opened the possibility that quiescent, G0/G1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G0/G1. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G0/G1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP+ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.

Abstract

To develop an antibody-based screen for epitopes involved in botryllid historecognition, BALB/c mice were immunized with whole Botryllus schlosseri colonies. Resulting monoclonal antibodies were screened for alpha or beta fusibility types using enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. One monoclonal antibody (109) that recognized a polymorphic epitope was further analyzed by Western blotting. It binds a species-specific epitope localized to the atrial siphon and blood vessels. The epitope does not cosegregate with fusibility type. A complementary DNA clone encoding this antigen contains an endoplasmic reticulum retention motif. Polymorphism observed on Western blots was confirmed by Northern blot analysis. This antigen provides a new polymorphic marker that may be useful in studies of tunic formation.

Abstract

Fas-deficient (Fas(lpr/lpr)) mice constitutively expressing Bcl-2 in myeloid cells by the hMRP8 promoter often develop a fatal disease analogous to human acute myeloblastic leukemia (AML-M2). Hematopoietic cells from leukemic Fas(lpr/lpr)hMRP8bcl-2 animals form clonogenic blast colonies in vitro and can transfer disease to wild-type mice. In vitro ligation of Fas on Fas+/+ hMRP8bcl-2 marrow cells depletes approximately 50% of myeloid progenitor activity, demonstrating that Bcl-2 can only partially block Fas-mediated death signals in myelomonocytic progenitors. In addition, Fas(lpr/lpr) marrow contains greatly increased numbers of myeloid colony-forming cells as compared to Fas+/+ controls. Taken together, these data suggest that Fas has a novel role in the regulation of myelopoiesis and that Fas may act as a tumor suppressor to control leukemogenic transformation in myeloid progenitor cells.

Abstract

In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.

Abstract

Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

Abstract

The protochordate allorecognition system has long invited comparison with the vertebrate major histocompatibility complex (MHC). In the colonial species Botryllus schlosseri, a rapid fusion or rejection response resembling graft acceptance or rejection in vertebrates is controlled by a single highly polymorphic genetic region. Because linkage between heat shock protein 70 (HSP70) genes and the MHC appears to be conserved within the vertebrate lineage, linkage relationships between two HSP70 genes (HSP70.1 and HSP70.2) and the historecognition locus (FuHC) have been analyzed in B. schlosseri. Segregation patterns of restriction fragment length polymorphisms located in the 3' flanking regions of HSP70.1 and HSP70.2 were determined for progeny of defined crosses. These progeny were also analyzed for fusibility type by an in vivo cut colony assay. No close linkage was detected between any of the three loci. These results do not support the hypothesis that the allorecognition response in B. schlosseri is determined by an MHC homologue. However, it remains a possibility that orthologues of other MHC-linked genes will be linked to the B. schlosseri FuHC.

Abstract

The colonial protochordate, Botryllus schlosseri, undergoes a genetically defined, natural transplantation reaction when the edges of two growing colonies interact. Peripheral blood vessels of each colony touch and will either fuse together to form a common vasculature between the colonies, or reject each other in an active blood-based inflammatory process in which the interacting vessels are cut off and the two colonies no longer interact. Previous studies have demonstrated that allorecognition in Botryllus is principally controlled by a single Mendelian locus named the fusion/histocompatibility (Fu/HC) locus, with multiple codominantly expressed alleles. However, identification and cloning of this locus has been difficult. We are taking a genomic approach in isolating this locus by creating a detailed genetic linkage map of the 725 Mbp Botryllus genome using DNA polymorphisms (primarily identified as AFLPs) as molecular genetic markers. DNA polymorphisms are identified in inbred laboratory strains of Fu/HC defined Botryllus, and their segregation and linkage is analyzed in a series of defined crosses. Using bulk segregant analysis, we have focused our mapping efforts on the Fu/HC region of the genome, and have generated an initial map which delineates the Fu/HC locus to a 5.5 cM region.

Abstract

A new transgenic mouse has been generated in which the proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2 transgenic mice overexpress BCL-2 in all cells of the hematolymphoid system and have been used to assess the role of BCL-2 in protecting cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-BCL-2 transgenic mice has two components: an increase in the radioresistance of individual cells and, to a lesser extent, an increase in the size of certain critically important cell populations, such as HSC. Bone marrow transplantation experiments show that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation is not limited to the increased expression levels of BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in lymphocyte populations that survive irradiation in wild-type mice. We show that ubiquitous overexpression of BCL-2 in the hematopoietic system can be used to increase the resistance of animals to lethal challenges such as irradiation.

Abstract

The Ikaros gene encodes multiple protein isoforms that contribute critical functions during the development of lymphocytes and other hematopoietic cell types. The intracellular functions of Ikaros are not known, although recent studies have shown that Ikaros proteins colocalize with inactive genes and centromeric heterochromatin. In this study, Ikaros proteins were found to be components of highly stable complexes. The complexes from an immature T cell line were purified, revealing associated proteins of 70 and 30 kD. The p70 gene, named Helios, encodes two protein isoforms with zinc finger domains exhibiting considerable homology to those within Ikaros proteins. Helios and Ikaros recognize similar DNA sequences and, when overexpressed, Helios associates indiscriminately with the various Ikaros isoforms. Although Ikaros is present in most hematopoietic cells, Helios was found primarily in T cells. The relevance of the Ikaros-Helios interaction in T cells is supported by the quantitative association of Helios with a fraction of the Ikaros. Interestingly, the Ikaros-Helios complexes localize to the centromeric regions of T cell nuclei, similar to the Ikaros localization previously observed in B cells. Unlike the B cell results, however, only a fraction of the Ikaros, presumably the fraction associated with Helios, exhibited centromeric localization in T cells. These results establish immunoaffinity chromatography as a useful method for identifying Ikaros partners and suggest that Helios is a limiting regulatory subunit for Ikaros within centromeric heterochromatin.

Two distinct pathways of positive selection for thymocytesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAAkashi, K., Kondo, M., Weissman, I. L.1998; 95 (5): 2486-2491

Abstract

Most mouse thymocytes undergoing positive selection are found on one of two pathways; the c-Kit+ and the c-Kit- pathways. Here, we show that c-Kit and interleukin-7 receptor (IL-7R)-mediated signals support positive selection during the transition from the subpopulation that first expresses cell surface T cell receptor (TCR)-the TCRalpha/betaloCD4(int)/CD8(int) (DPint) c-Kit+ cells to TCRalpha/betamedc-Kit+ transitional intermediate cells (the c-Kit+ pathway). Cells that fail positive selection on the c-Kit+ pathway become TCRalpha/betaloc-Kit- (DPhi) blasts that appear to undergo alternative TCRalpha rearrangements. The rare DPhic-Kit- blast cells that thus are salvaged for positive selection by expressing a self-major histocompatibility complex selectable TCRalpha/beta up-regulate IL-7R, but not c-Kit, and are the principal progenitors on the c-Kit- pathway; this c-Kit-IL-7R+ pathway is mainly CD4 lineage committed. Cell division is a feature of the TCRlo-medc-Kit+ transition, but is not essential for CD4 lineage maturation from DPhic-Kit- blasts. In this view, positive selection on the c-Kit- path results from a salvage of cells that failed positive selection on the c-Kit+ path.

Abstract

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

Abstract

Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs.BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days).All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9.These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.

Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKlug, C. A., Morrison, S. J., Masek, M., Hahm, K., Smale, S. T., Weissman, I. L.1998; 95 (2): 657-662

Abstract

The generation of lymphoid cells in mice depends on the function of the Ikaros protein. Ikaros has been characterized as a lymphoid-restricted, zinc-finger transcription factor that is derived from an alternatively spliced message. Ikaros knockout mice have defects in multiple cell lineages, raising the question of whether the protein regulates multiple committed progenitors and/or multipotent stem cells. To address this issue, we examined Ikaros expression in purified populations of multipotent cells and more committed progenitors. We found that the DNA-binding isoforms of Ikaros were localized in the nucleus of the most primitive hematopoietic stem cell subset. Changes in the RNA splicing pattern of Ikaros occurred at two stages: (i) as long-term self-renewing stem cells differentiated into short-term self-renewing stem cells and (ii) as non-self-renewing multipotent progenitors differentiated into lymphoid-committed progenitors. Unexpectedly, we found Ikaros localized to heterochromatin in Abelson-transformed pre-B lymphocytes by using immunogold electron microscopy. These observations suggest a complex role for Ikaros in lymphoid development.

Abstract

We have identified a population of cells in murine bone marrow that has many of the phenotypic characteristics attributed to resting hematopoietic stem cells but does not reconstitute irradiated mice. These cells express high levels of Sca-1, H-2K and CD38 and low levels of Thy-1.1, but do not express CD34 nor any of the lineage markers including CD3, CD4, CD5, CD8 NK1.1, I-A, B220, Ig(MGA), CD40, kappa, Mac-1, Gr-1 or Ter119. In addition, this population can be found at normal frequency in nu/nu as well as rag-1-/- mice. These cells incorporate only low levels of Rh123, are resistant to the cytotoxic effects of 5-fluorouracil and, consistent with their resting phenotype, less than 2% of these cells are in the S/G2/M phases of the cell cycle. The only phenotypic characteristic that distinguishes these cells from the lineage- Sca-1+, Thy-1.1low long-term reconstituting hematopoietic stem cell population is their lack of c-kit expression. Here we have explored the possibility that these cells represent a truly resting population of hematopoietic stem cells. We found that the lineage-, Sca-1+, c-kit- cells do not respond to hematopoietic growth factors in vitro, either alone or in combination with stromal layers. Furthermore, these cells do not form in vivo spleen colonies nor do they have the ability to reconstitute irradiated mice. Thus, this population may represent either a population of resting stem cells for which we lack the appropriate activating stimulus, or simply represent a "mystery population" that phenotypically mimics most of the physical properties of resting stem cells. Given the close phenotypic similarity of the c-kit- mystery population cells to the c-kit+ long-term reconstituting stem cells, investigators must be rigorous to exclude their effects from other stem cell assays.

Abstract

During fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules briefly express the Peyer's patch addressin MAdCAM-1. This allows initial seeding by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta 7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. It was found that the CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1. They can differentiate into natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. In addition to LN, CD4+CD3- cells can also be found in fetal spleen starting at 13.5 dpc, while absent from fetal liver. In view of the necessity of lymphotoxin in lymphoid organ development, it is thought that the novel subset of CD4+CD3- LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.

Abstract

The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1(lo)Lin-/loSca-1(+) (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

Abstract

The existence of a common lymphoid progenitor that can only give rise to T cells, B cells, and natural killer (NK) cells remains controversial and constitutes an important gap in the hematopoietic lineage maps. Here, we report that the Lin(-)IL-7R(+)Thy-1(-)Sca-1loc-Kit(lo) population from adult mouse bone marrow possessed a rapid lymphoid-restricted (T, B, and NK) reconstitution capacity in vivo but completely lacked myeloid differentiation potential either in vivo or in vitro. A single Lin(-)IL-7R(+)Thy-1(-)Sca-1loc-Kit(lo) cell could generate at least both T and B cells. These data provide direct evidence for the existence of common lymphoid progenitors in sites of early hematopoiesis.

Abstract

For a brief period during fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules surprisingly express the Peyer's patch addressin MAdCAM-1. This expression allows initial seeding of this incipient structure by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. We show here that CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1 and that can become natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. Since the necessity of lymphotoxin in lymphoid organ development has been shown, we propose that the novel subset of CD4+CD3-LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.

Abstract

Five mircosatellite loci are characterized for the colonial ascidian Botryllus schlosseri. Within one population from Monterey, California, these loci have 3 to 17 alleles, observed heterozygosities from 0.40 to 0.63, expected heterozygosities from 0.52 to 0.84, and an overall paternity exclusion rate (QT) of 0.78. Three of the five loci demonstrated Mendelian patterns of inheritance in laboratory crosses. The size distribution of alleles suggests that most allelic diversity within these loci is generated by single-step and less frequently multistep mutations. However, several alleles may also have been generated by single based insertions or deletions. Mutation rates for the five microsatellite loci are less than 1 x 10(-2) per generation. Because or their highly polymorphic nature, these loci should prove useful for exploring issues of identity, kinship, population structure, and phylogenetics.

Abstract

The common cytokine receptor gamma chain (gamma(c)) is an indispensable subunit for the formation of lymphoid-related cytokine receptors, including IL-7 and IL-15 receptors, that mediate nonredundant or critical signals for the differentiation of T and B cells and natural killer (NK) cells, respectively. We introduced the bcl-2 transgene driven by E mu or H-2K promoters into gamma(c)-deficient mice that lack all three lymphoid subclasses. The forced expression of Bcl-2 restored all stages of T lymphopoiesis, but not B or NK cell development, indicating that a primary function of gamma(c)-mediated signals in the T lineage might be to maintain cell survival. Therefore, the development of T, B, and NK cells may be influenced by distinct intracytoplasmic signaling cascades that are activated by coupling of gamma(c)-related receptors.

Abstract

Osteopetrotic (op/op) mice lack functional M-CSF and have depressed levels of macrophages and osteoclasts. We prepared transgenic mice (hMRP8bcl-2) that express human Bcl-2 in monocytes. In vitro hMRP8bcl-2 monocytes do not undergo apoptosis in the absence of serum and M-CSF, while op/op and wild-type monocytes die. These Bcl-2-expressing monocytes spontaneously undergo macrophage differentiation. In vivo, the op/op hMRP8bcl-2 mice show significant replenishment of tissue macrophages. Their long bone osteopetrosis is largely reversed, and extensive medullary hematopoiesis appears in the bone marrow. We propose that M-CSF augments monocyte survival, permitting them to respond to internal and external cues for their differentiation.

Abstract

Mice lacking functional IL-7 or IL-7R alpha genes are severely deficient in developing thymocytes, T cells, and B cells. IL-7 and IL-7 receptor functions are believed to result in lymphoid cell proliferation and cell maturation, implying signal transduction pathways directly involved in mitogenesis and elaboration of developmentally specific new gene programs. Here, we show that enforced expression of the bcl-2 gene in T-lymphoid cells (by crossing in the Emu-bcl-2 transgene) in IL-7R alpha-deficient mice results in a significant restoration of thymic positive selection and T cell numbers and function. We propose cell survival signals to be the principal function of IL-7R engagement in thymic and T cell development.

Abstract

We review the development of the hematopoietic system, focusing on the transition from hematopoietic stem cells (HSCs) to T cells. This includes the isolation of HSCs, and recent progress in understanding their ontogeny, homing properties, and differentiation. HSC transplantation is reviewed, including the kinetics of reconstitution, engraftment across histocompatibility barriers, the facilitation of allogeneic engraftment, and the mechanisms of graft rejection. We describe progress in understanding T-cell development in the bone marrow and thymus as well as the establishment of lymph nodes. Finally, the role of bcl-2 in regulating homeostasis in the hematopoietic system is discussed.

Abstract

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU-enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU-treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

Abstract

All multipotent hematopoietic progenitors in C57BL-Thy-1.1 bone marrow are divided among three subpopulations of Thy-1.1(lo) Sca-1+ Lin(-/lo) c-kit+ cells: long-term reconstituting Mac-1- CD4- c-kit+ cells and transiently reconstituting Mac-1(lo) CD4- or Mac-1(lo) CD4(lo) cells. This study shows that the same populations, with similar functional activities, exist in mice whose hematopoietic systems were reconstituted by hematopoietic stem cells after lethal irradiation. We demonstrate that these populations form a lineage of multipotent progenitors from long-term self-renewing stem cells to the most mature multipotent progenitor population. In reconstituted mice, Mac-1- CD4- c-kit+ cells gave rise to Mac-1(lo) CD4- cells, which gave rise to Mac-1(lo) CD4(lo) cells. Mac-1- CD4- c-kit+ cells had long-term self-renewal potential, with each cell being capable of giving rise to more than 10(4) functionally similar Mac-1- CD4- c-kit+ cells. At least half of Mac-1(lo) CD4- cells had transient self-renewal potential, detected in the spleen 7 days after reconstitution. Mac-1(lo) CD4(lo) cells did not have detectable self-renewal potential. The identification of a lineage of multipotent progenitors provides an important tool for identifying genes that regulate self-renewal and lineage commitment.

Abstract

The past year provided a number of challenges to our expectations regarding hematopoietic stem cell (HSC) biology. Evidence has emerged that HSCs arise intraembryonically before they can be detected in the yolk sac. A number of genes that may regulate the formation, self-renewal, or differentiation of HSC have been identified. New markers for purifying HSCs have also been described. Although different groups have attributed different properties to HSCs, it now appears that the differences may be explained by variations in assay conditions rather than by differences in the HSCs themselves. Finally, insights have emerged into the complexity of the regulation of HSC proliferation and adhesion properties.

Abstract

The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.

Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilizationPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMorrison, S. J., Wright, D. E., Weissman, I. L.1997; 94 (5): 1908-1913

Abstract

We isolated hematopoietic stem cells (HSC) from mice treated with cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). All mobilized multipotent progenitor activity was contained in two populations: Thy-1(lo) Sca-1+ Lin- Mac-1- CD4- c-kit+ long-term reconstituting progenitors and Thy-1(lo) Sca-1+ Lin- Mac-1(lo) CD4- transiently reconstituting progenitors. CY/G-CSF treatment drove both long-term and transient multipotent progenitors into cycle, leading to a more than 12-fold expansion in the number of long-term self-renewing HSC prior to mobilization. After CY and 2 days of G-CSF treatment the number of bone marrow HSC began to decline and the number of blood and splenic HSC increased. HSC continued to proliferate in the bone marrow and spleen through 8 days of G-CSF treatment, but HSC released into the blood tended to be in G0/G1 phase. Mobilized multipotent progenitors isolated from the spleen were less efficient than normal bone marrow multipotent progenitors in engrafting irradiated mice but did not differ in colony forming unit-spleen (CFU-S) activity or single cell in vitro assays of primitive progenitor activity. The data suggest that mobilized HSC isolated from the spleen are less efficient at homing to and engrafting the bone marrow of irradiated recipient mice.

Abstract

The colonial protochordate Botryllus schlosseri possesses a historecognition system which has long invited comparison to the vertebrate MHC. Upon contact, colonies either fuse or reject one another in a manner resembling graft acceptance or rejection in vertebrates. This response is controlled by a single highly polymorphic genetic region, the FuHC locus. Colonial protochordates such as B. schlosseri are among the closest relatives of the vertebrate lineage, and therefore may possess a recognizable MHC homologue. Since linkage between heat shock protein 70 (HSP70) genes and MHC appears to be conserved within the vertebrate lineage, we have analyzed HSP70 genes from B. schlosseri as a first step toward isolating the historecognition locus. Two HSP70 genes (HSP70.1 and HSP70.2) have been cloned and sequenced, and exhibit 93.6% sequence identity within the predicted coding regions. The B. schlosseri genes share a number of characteristics with vertebrate MHC-linked HSP70 genes: Northern blotting and sequence analysis suggest that the protochordate genes are cytoplasmically-expressed heat-inducible members of the HSP70 gene family (FAGAN and WEISSMAN 1996). However, unlike vertebrate MHC-linked HSP70 genes, HSP70.1 and HSP70.2 are not closely linked (FAGAN and WEISSMAN 1997). Furthermore, neither is closely linked to the locus determining historecognition (FAGAN and WEISSMAN 1997). These results do not support the hypothesis that the B. schlosseri FuHC locus is an MHC homolog. A discussion of the implications of these results for evolution of the vertebrate MHC is included.

Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition systemPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAStoner, D. S., Weissman, I. L.1996; 93 (26): 15254-15259

Abstract

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell "parasitism" (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337-5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss's hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

Abstract

Flow cytometry and cell sorting have been employed in order to identify and purify murine neutrophils and monocytes. Using a combination of antibodies, we were able to distinguish between these two subsets of myeloid cells. The method described in this paper is simple to perform and can be applied to characterize myeloid cells from blood, spleen, bone marrow and after an induced inflammation.

Abstract

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

Abstract

We have purified hematopoietic stem cells (HSCs) from the bone marrow of old mice and compared their properties to HSCs in young and middle-aged mice. Single, reconstituting HSCs (by limit dilution) from old and young mice exhibited indistinguishable progenitor activities in vivo. HSCs were five times as frequent in the bone marrow of old mice; however, HSCs from old mice were only one-quarter as efficient at homing to and engrafting the bone marrow of irradiated recipients. HSCs in young and middle-aged mice rarely were in the S/G2/M phases of the cell cycle, but HSCs in old mice were frequently in cycle. We speculate that the unexpected proliferation of HSCs in old mice might be related to the increased incidence of leukemia in old mice. HSCs change with age, but it is unknown whether these changes are determined intrinsically or caused by the aging of their environment.

Abstract

It has been proposed that the biological clock underlying the limited division potential of eukaryotic cells is telomere length. We assayed telomerase activity in single cells of the hematopoietic and immune systems. We examined hematopoietic stem cells at four stages of differentiation, lineage-committed progenitors, and mature myeloid and lymphoid cells. The frequency of telomerase-expressing cells within each population was proportional to the frequency of cells thought to have self-renewal potential. Among bone marrow hematopoietic stem cells, 70% exhibited detectable telomerase activity. The telomerase-expressing somatic cells observed in this study are not thought to be immortal, and expression was not correlated with cell cycle distribution or differentiation state. This study demonstrates that the developmental characteristic most consistently associated with telomerase expression is self-renewal potential.

Abstract

CD3-4-8- interleukin-2 receptor positive (IL-2R+) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R+CD3-4-8- thymocytes were obtained by positive panning of IL-2R+ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2 1/2 days significant numbers of CD4+8+ and CD3+4+8- cell populations developed, the latter expressing the alpha beta T-cell receptor (alpha beta-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R+ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.

Abstract

Cell death is an important physiological process, but it can be triggered by both physiological and nonphysiological stimuli. The product of the bcl-2 gene has the ability to inhibit a physiological cell death process that can be activated by a variety of physiological signals, such as growth factor deprivation. This report describes the use of electron microscopy to examine the effects of two cytotoxic drugs on factor-dependent cells that constitutively express the human bcl-2 gene. Although all cells treated with sodium azide showed changes typical of necrosis, in the absence of Bcl-2 the cells died more rapidly and also displayed features of apoptosis. The fact that Bcl-2 could delay cell death argues that cells can activate internal cell death mechanisms to commit suicide before they are killed by a cytotoxin. Northern analysis showed that growth factor did not preserve viability of the cells through induction of bcl-2. However, growth factor may prevent activation of the physiological cell death mechanisms that bcl-2 can control. This process may constitute a primitive defense response, and blocking it may provide a means of limiting damage caused by otherwise sublethal injuries.

Abstract

IDDM is a chronic inflammatory disease in which there is autoimmune-mediated organ-specific destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The migration of autoreactive lymphocytes and other leukocytes from the bloodstream into the target organ is of clear importance in the etiology of many organ-specific autoimmune/inflammatory disorders, including IDDM. In IDDM, this migration results in lymphocytic invasion of the islets (formation of insulitis) and subsequent destruction of beta-cells. Migration of lymphocytes from the bloodstream into tissues is a complex process involving sequential adhesion and activation events. This migration is controlled in part by selective expression and functional regulation of cell adhesion molecules (CAMs) on the surface of lymphocytes and vascular endothelial cells or in the extracellular matrix. Understanding the mechanisms that regulate lymphocyte migration to the pancreatic islets will lead to further understanding of the pathogenesis of IDDM. In this article, we summarize the recent advances regarding the function of CAMs in the development of IDDM in animal models and in humans and discuss the potential for developing CAM-based therapies for IDDM.

Abstract

TCR gene rearrangement is strictly regulated during mouse ontogeny. The V-(D)-J junctions of alphabeta and gammadelta TCR transcripts expressed in the adult thymus are more highly diverse than those in the fetal thymus. We previously showed that adult hematopoietic stem cells (HSC) have a higher capacity to insert N nucleotides into Vgamma4 TCR transcripts than fetal HSC and that the level of N nucleotide insertion is determined, at least in part, at the level of HSC. To analyze this developmental change of HSC at the single cell level, we investigated N nucleotide insertions in three TCR transcripts (Vgamma4, Vgamma2 and Vbeta8) derived from limiting numbers of fetal liver HSC by fetal thymic organ culture. Eight day-14 fetal liver HSC clones showed various levels of N nucleotide insertions in Vgamma transcripts (0-78%). On the other hand, the level of N insertions was similarly regulated in Vgamma4, Vgamma2, and Vbeta8 TCR transcripts in a clone-specific way. These results suggested that the level of N insertion is programmed at the level of single HSC and that fetal liver contains a heterogeneous population of HSC in terms of N insertion capacity. After 3 weeks culture with a stromal cell line, fetal HSC showed higher levels of N insertion capacity than before culture. This result and the presence of HSC with intermediate N insertion capacity support the hypothesis that the developmental potential of individual HSC gradually changes from fetal to adult type in one stem cell lineage.

Abstract

Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele.

Abstract

Mouse hematopoietic stem cells (HSCs) are highly enriched in the rare (approximately 0.05%) Thy-1.1(lo)Lin(-/lo)Sca-1+ fraction of hematopoietic tissues. It has been demonstrated that Thy-1.1(lo)Lin(-/lo)Sca-1+ cells are the only HSCs in C57BL/Ka-Thy-1.1 bone marrow. In this study, we separated C57/Ka-Thy-1.1 bone marrow cells by counterflow centrifugal elutriation (CCE) into four fractions and characterized Thy-1.1(lo)Lin(-/lo)Sca-1+ cells in each eluted fraction. The peak number of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells was highly enriched in one eluted fraction, which was also highly enriched for day-12 to -13 CFU-S. Activities for day-13 CFU-S, radioprotection, and long-term multilineage reconstitution correlated with, and could be generally predicted by determining, the frequency of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells in a given eluted fraction. However, the fraction that was highly enriched for blast cells and contained a low frequency of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells, only provided short-term but not long-term radioprotection, with a predicted cell number (100 cells) that should have protected > or = 95% (PD95) of hosts. Still, when 300 Thy-1.1(lo)Lin(-/lo)Sca-1+ cells from this fraction (3 X PD95 for unfractionated HSCs) were injected, mice were radioprotected and donor cells provided long-term multilineage reconstitution. We propose that such blast cells may contain two Thy-1.1(lo)Lin(-/lo)Sca-1+ subsets, one providing short-term and the other long-term multilineage sustained hematopoiesis, the latter presumably due to HSC self-renewal.